KR100607610B1 - Compositions and methods for the diagnosis and treatment of tumor - Google Patents

Abstract

The present invention relates to a composition comprising a substance useful for the diagnosis and treatment of a tumor in a mammal, and to a method for diagnosing and treating the tumor using the composition.

Tumors, tumor-associated antigenic targets (TATs), antibodies.

Description

Method for the diagnosis and treatment of tumors and compositions therefor {COMPOSITIONS AND METHODS FOR THE DIAGNOSIS AND TREATMENT OF TUMOR}

1 is the nucleotide sequence of TAT169 cDNA (SEQ ID NO: 1) and SEQ ID NO: 1 is a clone referred to herein as “DNA71290-1630”.

2 is the nucleotide sequence of TAT170 cDNA (SEQ ID NO: 2), wherein SEQ ID NO: 2 is a clone referred to herein as "DNA76393-1664".

3 is the nucleotide sequence of TAT171 cDNA (SEQ ID NO: 3), wherein SEQ ID NO: 3 is a clone referred to herein as "DNA53971-1359".

4 is the nucleotide sequence of TAT172 cDNA (SEQ ID NO: 4), and SEQ ID NO: 4 is a clone referred to herein as “DNA56439-1376”.

5 is the nucleotide sequence of TAT173 cDNA (SEQ ID NO: 5), wherein SEQ ID NO: 5 is a clone referred to herein as “DNA64852-1589”.

FIG. 6 shows an amino acid sequence (SEQ ID NO: 6) derived from the coding sequence of SEQ ID NO: 1 shown in FIG.

FIG. 7 shows an amino acid sequence derived from the coding sequence of SEQ ID NO: 2 shown in FIG. 2 (SEQ ID NO: 7).

FIG. 8 shows the amino acid sequence (SEQ ID NO: 8) derived from the coding sequence of SEQ ID NO: 3 shown in FIG.

9 shows an amino acid sequence (SEQ ID NO: 9) derived from the coding sequence of SEQ ID NO: 4 shown in FIG.

FIG. 10 shows an amino acid sequence (SEQ ID NO: 10) derived from the coding sequence of SEQ ID NO: 5 shown in FIG.

The present invention relates to compositions of matter useful for the diagnosis and treatment of tumors in mammals and methods of using the compositions.

Malignant tumors (cancer) are the second leading cause of death after heart disease in the United States (Boring et al., CA Cancer J. Clin., 43: 7 (1993)). Cancer is an increase in the number of abnormal or neoplastic cells (proliferating to form tumor masses) derived from normal tissue, infiltrating into adjacent tissue by these neoplastic tumor cells, and through a blood or lymphatic system in a process called ex In fact, it is characterized by the development of malignant cells that spread to local lymph nodes and distant sites. Cancerous cells proliferate under conditions that would not grow if they were normal. The cancer itself appears in a wide variety of forms, characterized by different degrees of invasiveness and invasiveness.

In an attempt to find cell targets effective for cancer therapy, researchers sought to identify polypeptides that specifically overexpress one or more normal non-cancerous cell (s) on certain types of cancer cell surfaces. The identification of such tumor-associated cell surface antigen polypeptides allowed the cancer cells to be specifically targeted and destroyed via antibody-based therapy. In this regard, it should be understood that antibody-based therapies have proven to be very effective in the treatment of certain cancers. For example, HERCEPTIN® and RITUXAN® (Genentech, Inc., South San Francisco, CA, USA) treat breast cancer and non-Hodgkin lymphomas, respectively. It is an antibody that has been used successfully. More specifically, HERCEPTIN® is a humanized monoclonal antibody derived from recombinant DNA that selectively binds to the extracellular domain of the human epidermal growth factor receptor 2 (HER2) oncogene. Overexpression of HER2 protein is observed in 25-30% of primary breast cancers. RITUXAN® is a genetically engineered chimeric murine / human monoclonal antibody directed against CD20 antigen found on the surface of normal B lymphocytes and malignant B lymphocytes. Both of these antibodies are produced recombinantly in CHO cells.

In other attempts to find effective cell targets for cancer therapy, researchers produce and secrete by certain types of cancer cells at higher expression levels than those produced and secreted by one or more normal non-cancerous cell (s). To identify the resulting polypeptide. Often, these secretion factors are proteins that grow cancer cells more than normal cells, such as angiogenesis factors, cell adhesion factors, and growth factors. Identifying antagonists against these secreted polypeptides is expected to serve to provide effective therapeutics for the treatment of the cancer. In addition, identification of such overexpression of secretion factors will be useful for the diagnosis of certain cancers in mammals.

However, despite these advances in mammalian cancer therapy, there is a high demand for additional diagnostic agents capable of detecting the presence of tumors in mammals and therapeutic agents that effectively inhibit neoplastic cell growth, respectively. Accordingly, it is an object of the present invention to identify cell surface polypeptides overexpressed on normal cancer cells or other different cancer cells, or secreted polypeptides overexpressed by certain cancer cells relative to normal cells or other cancer cells, and such polypeptides and these It is to prepare a composition useful for the therapeutic treatment and diagnostic detection of cancer in a mammal using the coding nucleic acid of.

Summary of the Invention

A. Embodiment

Herein, Applicants first relate to the identification of various cell polypeptides (and coding nucleic acids or fragments thereof) that are expressed at a higher degree on the surface of one or more types of cancer cells compared to one or more types of normal non-cancer cells. List it. Such polypeptides are referred to herein as tumor-associated Antigenic Target polypeptides (“TAT” polypeptides), which are expected to function as effective targets for cancer therapy and diagnosis in mammals.

Accordingly, one embodiment of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a tumor-associated antigenic target polypeptide or fragment thereof (“TAT” polypeptide).

In certain aspects, the isolated nucleic acid molecule is (a) a full-length TAT polypeptide having an amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence without a signal peptide as disclosed herein, with or without a signal peptide as described herein. DNA molecules encoding the extracellular domain of the transmembrane TAT polypeptide or any other specially defined fragment of the full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) a nucleic acid with the complement of said DNA molecule (a) At least about 80%, alternatively about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Nucleotide sequences that are at least 94%, 95%, 96%, 97%, 98%, or 99%.

In another aspect, the isolated nucleic acid molecule comprises (a) the coding sequence of the full-length TAT polypeptide cDNA as disclosed herein, the coding sequence of the TAT polypeptide without a signal peptide as disclosed herein, or the signal peptide as described herein, or A DNA molecule comprising a coding sequence of an extracellular domain of a transmembrane TAT polypeptide that is absent or of any other specially defined fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) said DNA molecule (a ) Nucleic acid sequence identity with the complement of at least about 80%, alternatively about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% At least 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

In a further aspect, the present invention provides a DNA molecule encoding the same mature polypeptide as that encoded by (a) the full-length coding sequence of any human protein cDNA deposited with the ATCC, or (b) said DNA molecule. nucleic acid sequence identity with the complement of (a) is at least about 80%, alternatively about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, An isolated nucleic acid molecule comprising a nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In this regard, the term “full length coding sequence” refers to the TAT polypeptide-encoding nucleotide sequence of cDNA inserted in a vector deposited in the ATCC, which in the accompanying figures is often included between the start codon and the stop codon.

Another aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a TAT polypeptide from which a transmembrane domain has been deleted or in which a transmembrane domain has been inactivated or a nucleotide sequence complementary to such a coding nucleotide sequence. Transmembrane domain (s) of the s) are disclosed herein. Thus, soluble extracellular domains of the TAT polypeptides described herein are contemplated.

In another aspect, the invention provides an antibody comprising (a) a TAT polypeptide having a full-length amino acid as disclosed herein, a TAT polypeptide amino acid sequence without a signal peptide as disclosed herein, and a transmembrane TAT polypeptide with or without a signal peptide as described herein. An nucleotide sequence encoding the extracellular domain of or any other specially defined fragment of the full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) an isolated nucleic acid molecule that hybridizes with the complement of said nucleotide sequence (a) It is about. In this regard, one embodiment of the invention comprises a binding site for, for example, a diagnostic probe, an antisense oligonucleotide probe or optionally an anti-TAT polypeptide antibody, a TAT binding oligopeptide or another organic small molecule that binds to a TAT polypeptide. A fragment of the full-length TAT polypeptide-encoding sequence as described herein, or a complement thereof, which can be used as a hybridization probe or the like useful for a coding fragment of a full-length TAT polypeptide capable of encoding a polypeptide. Typically, such nucleic acid fragments are at least about 5 nucleotides in length, alternatively about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 , 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220 , 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 , 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720 , 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950 , 960, 970, 980, 990 or 1000 or more, wherein the term "about" in this context means 10% of the nucleotide sequence length ± this length. New fragments of TAT polypeptide-encoding nucleotide sequences can be used to align the TAT polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well-known sequence alignment programs, and to identify any TAT polypeptide-encoding nucleotide sequence fragments. It is to be understood that the determination may be made in a routine manner by determining if the (s) are new. All such novel fragments of TAT polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are TAT polypeptide fragments encoded by these nucleotide molecule fragments, preferably TAT polypeptide fragments comprising binding sites for anti-TAT antibodies, TAT binding oligopeptides or other organic small molecules that bind to TAT polypeptides.

In another embodiment, the present invention provides an isolated TAT polypeptide encoded by any isolated nucleic acid sequence identified above.

In certain aspects, the invention provides a cell of a TAT polypeptide having a full length amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence without a signal peptide as disclosed herein, or a transmembrane TAT polypeptide with or without a signal peptide as described herein. At least about 80%, or alternatively, about 100% amino acid sequence identity to an extradomain, an amino acid sequence encoded by any nucleic acid sequence disclosed herein, or any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% And an isolated TAT polypeptide comprising an amino acid sequence that is at least 98% or 99%.

In a further aspect, the invention provides an amino acid sequence identity of at least about 80%, alternatively about 81%, 82%, 83% with an amino acid sequence encoded by any human protein cDNA deposited with ATCC as disclosed herein , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% It relates to an isolated TAT polypeptide comprising a sequence.

In a particular aspect, the invention provides an isolated TAT polypeptide free of N-terminal signal peptide and / or initiating methionine as described herein and encoded by a nucleotide sequence encoding said amino acid sequence. Also described herein is a method of making an isolated TAT polypeptide, which method comprises culturing a host cell comprising a vector comprising a suitable coding nucleic acid molecule under conditions suitable for expression of the TAT polypeptide, and culturing the cell. Recovering the TAT polypeptide from water.

Another aspect of the invention provides an isolated TAT polypeptide wherein the transmembrane domain is deleted or the transmembrane domain is inactivated. Also described herein is a method of making an isolated TAT polypeptide, which method comprises culturing a host cell comprising a vector comprising a suitable coding nucleic acid molecule under conditions suitable for expression of the TAT polypeptide, and culturing the cell. Recovering the TAT polypeptide from water.

In another embodiment of the invention, the invention provides a vector comprising a DNA encoding any of the polypeptides described herein. Also provided are host cells comprising any of these vectors. For example, host cells are CHO cells, E. coli. E. coli or yeast. Also provided is a method of making any of the polypeptides described herein, the method comprising culturing the host cell under conditions suitable for expression of the desired polypeptide, and recovering the desired polypeptide from the cell culture. .

In other embodiments, the invention provides an isolated chimeric polypeptide comprising any of the TAT polypeptides described herein fused to a heterologous (non-TAT) polypeptide. Examples of such chimeric molecules include any TAT polypeptide described herein fused to a heterologous polypeptide, such as an epitope tag sequence or an Fc region of an immunoglobulin.

In other embodiments, the invention provides antibodies that preferably bind specifically to any polypeptide described above or below. If desired, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody or single-chain antibody. Antibodies of the invention may optionally be conjugated with growth inhibitors or cytotoxic agents such as toxins such as maytansinoids or calicheamicins, antibiotics, radioisotopes, nucleases, and the like. Antibodies of the invention can optionally be produced in CHO cells or bacterial cells, preferably inducing the death of the cells to which they bind. For diagnostic purposes, the antibodies of the invention may be detectably labeled or attached to a solid support or the like.

In another embodiment of the invention, the invention provides a vector comprising a DNA encoding any of the antibodies described herein. Also provided are host cells comprising any of these vectors. For example, host cells are CHO cells, E. coli. It may be coli or yeast. Also provided is a method of making any of the antibodies described herein, the method comprising culturing the host cell under conditions suitable for expression of the desired antibody, and recovering the desired antibody from the cell culture. .

In another embodiment, the present invention provides oligopeptides ("TAT binding oligopeptides") that preferably specifically bind to TAT polypeptides described above or below. In some cases, the TAT binding oligopeptides of the present invention may be conjugated with growth inhibitors or cytotoxic agents such as toxins such as maytansinoids or calicheamicins, antibiotics, radioisotopes, nucleases, and the like. TAT binding oligopeptides of the invention can optionally be produced in CHO cells or bacterial cells, preferably inducing the death of the cells to which they bind. For diagnostic purposes, the TAT binding oligopeptides of the invention may be detectably labeled or attached to a solid support or the like.

In another embodiment of the invention, the invention provides a vector comprising a DNA encoding any of the TAT binding oligopeptides described herein. Also provided are host cells comprising any of these vectors. For example, host cells are CHO cells, E. coli. It may be coli or yeast. Also provided is a method of preparing any of the TAT binding oligopeptides described herein, which method comprises culturing the host cell under conditions suitable for the expression of the desired oligopeptide, and recovering the desired oligopeptide from the cell culture. It includes a step.

In another embodiment, the present invention provides organic small molecules ("TAT binding organic molecules") that preferably specifically bind to TAT polypeptides described above or below. In some cases, the TAT binding organic molecules of the present invention may be conjugated with growth inhibitors or cytotoxic agents such as toxins such as maytansinoids or calicheamicins, antibiotics, radioisotopes, nucleases, and the like. The TAT binding organic molecules of the invention preferably induce the death of the cells to which they bind. For diagnostic purposes, the TAT binding organic molecules of the invention can be detectably labeled or attached to a solid support or the like.

In a further embodiment, the invention provides a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT antibody as described herein, a TAT binding oligopeptide as described herein or It relates to a composition comprising a TAT binding organic molecule as described herein and a carrier. In some cases, the carrier is a pharmaceutically acceptable carrier.

In another embodiment, the present invention is directed to an article of manufacture comprising a container and a composition contained within the container, wherein the composition is a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, described herein Anti-TAT antibodies as described above, TAT binding oligopeptides as described herein or TAT binding organic molecules as described herein. In addition, the article may optionally have a label affixed to the container or a package insert contained within the container indicating that the composition is used for therapeutic treatment or diagnostic detection of the tumor.

Other embodiments of the invention include a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT antibody as described herein, a TAT binding oligopeptide as described herein or TAT polypeptide as described herein, chimeric TAT polypeptide as described herein, anti-TAT as described herein for the manufacture of a medicament useful for the treatment of a condition responsive to a TAT binding organic molecule as described. An antibody, a TAT binding oligopeptide as described herein, or a TAT binding organic molecule as described herein is directed to.

B. Additional Embodiments

Another embodiment of the invention is directed to a method of killing cancer cells expressing a TAT polypeptide, the method comprising contacting the cancer cells with an antibody, oligopeptide or organic small molecule that binds to the TAT polypeptide and eventually contacting the cancer cells. Kills. If desired, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules used in the methods of the invention may optionally contain growth inhibitors or cytotoxic agents such as toxins, antibiotics, radioisotopes, such as maytansinoids or calicheamicins, Nucleolytic enzymes and the like. Antibodies and TAT binding oligopeptides used in the methods of the invention may optionally be produced in CHO cells or bacterial cells.

Another embodiment of the invention relates to a method of therapeutically treating a TAT polypeptide-expressing tumor in a mammal, the method comprising administering to the mammal a therapeutically effective amount of an antibody, oligopeptide or organic small molecule that binds to the TAT polypeptide. And eventually treat the tumor effectively therapeutically. If desired, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules used in the methods of the invention may optionally contain growth inhibitors or cytotoxic agents such as toxins, antibiotics, radioisotopes, such as maytansinoids or calicheamicins, Nucleolytic enzymes and the like. Antibodies and oligopeptides used in the methods of the invention may optionally be produced in CHO cells or bacterial cells.

Another embodiment of the invention relates to a method of measuring the presence of a TAT polypeptide in a sample suspected of containing a TAT polypeptide, which method comprises exposing the sample to an antibody, oligopeptide or organic small molecule that binds to the TAT polypeptide. And measuring the binding of the antibody, oligopeptide or organic molecule with the TAT polypeptide in the sample, wherein the presence of this binding indicates the presence of the TAT polypeptide in the sample. In some cases, the sample may contain cells (which may be cancer cells) suspected of expressing a TAT polypeptide. Antibodies, TAT binding oligopeptides or TAT binding organic molecules used in the methods of the invention may optionally be detectably labeled, or may be attached to a solid support or the like.

A further embodiment of the invention relates to a method of diagnosing the presence of a tumor in a mammal, the method comprising (a) a test sample of tissue cells obtained from said mammal of a gene encoding a TAT polypeptide, and (b) Detecting a level of expression in a control sample of known normal cells originating from the same tissue, wherein the level of expression of the TAT polypeptide in the test sample is higher than that of the control sample to obtain the test sample. In mammals indicate the presence of a tumor.

Another embodiment of the invention relates to a method for diagnosing the presence of a tumor in a mammal, the method comprising: (a) an antibody, oligopeptide or organic small molecule that binds a test sample of tissue cells obtained from the mammal to a TAT polypeptide; Contacting, and (b) detecting complex formation between the antibody, oligopeptide or organic small molecule and the TAT polypeptide in the test sample, wherein the complex formation indicates the presence of a tumor in the mammal. In some cases, the antibody, TAT binding oligopeptide or TAT binding organic molecule used is detectably labeled, and / or attached to a solid support, and / or a test sample of tissue cells from an individual suspected of having a cancerous tumor. Get

C. Other Additional Embodiments

The following are other further embodiments claimed in the present invention:

<First Embodiment>

(a) a nucleotide sequence encoding the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10);

(b) encoding the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. Nucleotide sequence;

(c) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10). Nucleotide sequences that encode for;

(d) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. Nucleotide sequences that encode for;

(e) the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5);

(f) the full length coding sequence of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5);

(g) the full length coding sequence of the cDNA deposited with any ATCC accession number shown in Table 7; or

(h) the complement of (a), (b), (c), (d), (e), (f) or (g)

An isolated nucleic acid having at least 80% nucleic acid sequence identity with.

Second Embodiment

(a) a nucleotide sequence encoding the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10);

(b) encoding the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. Nucleotide sequence;

(c) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10). Nucleotide sequences that encode for;

(d) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. Nucleotide sequences that encode for;

(e) the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5);

(f) the full length coding sequence of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5);

(g) the full length coding sequence of the cDNA deposited with any ATCC accession number shown in Table 7; or

(h) the complement of (a), (b), (c), (d), (e), (f) or (g)

Isolated nucleic acid comprising a.

Third Embodiment

(a) a nucleotide sequence encoding the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10);

(b) encoding the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. Nucleotide sequence;

(c) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10). Nucleotide sequences that encode for;

(d) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. Nucleotide sequences that encode for;

(e) the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5);

(f) the full length coding sequence of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5);

(g) the full length coding sequence of the cDNA deposited with any ATCC accession number shown in Table 7; or

(h) the complement of (a), (b), (c), (d), (e), (f) or (g)

Isolated nucleic acid hybridizing with.

Fourth aspect

The nucleic acid of claim 3, wherein hybridization occurs under stringent conditions.

<Fifth aspect>

The nucleic acid of claim 3, wherein the nucleic acid is at least about 5 nucleotides in length.

<Sixth aspect>

An expression vector comprising the nucleic acid of the first aspect.

<Seventh aspect>

The expression vector of claim 6, wherein the nucleic acid is operably linked to a regulatory sequence recognized by the host cell transformed with the vector.

<Eighth aspect>

A host cell comprising the expression vector of the seventh aspect.

<9th aspect>

The CHO cell of claim 8, wherein the CHO cell is E. coli. Host cells that are E. coli cells or yeast cells.

<10th aspect>

Culturing the host cell of the eighth embodiment under conditions suitable for expression of the polypeptide, and recovering the polypeptide from the cell culture.

<Eleventh aspect>

(a) the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10);

(b) the amino acid sequence shown in Figure 6 (SEQ ID NO: 6), Figure 7 (SEQ ID NO: 7), Figure 8 (SEQ ID NO: 8), Figure 9 (SEQ ID NO: 9), or Figure 10 (SEQ ID NO: 10) without a linked signal peptide;

(c) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10). Amino acid sequence of;

(d) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. Amino acid sequence of;

(e) an amino acid sequence encoded by the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5);

(f) an amino acid encoded by the full length coding sequence of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5). order; or

(g) the amino acid sequence encoded by the full length coding sequence of the cDNA deposited with any ATCC accession number shown in Table 7

An isolated polypeptide having an amino acid sequence identity of 80% or more.

<Twelfth aspect>

(a) the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10);

(b) the amino acid sequence shown in Figure 6 (SEQ ID NO: 6), Figure 7 (SEQ ID NO: 7), Figure 8 (SEQ ID NO: 8), Figure 9 (SEQ ID NO: 9), or Figure 10 (SEQ ID NO: 10) without a linked signal peptide;

(c) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10). Amino acid sequence of;

(d) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. Amino acid sequence of;

(e) an amino acid sequence encoded by the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5);

(f) an amino acid encoded by the full length coding sequence of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5). order; or

(g) the amino acid sequence encoded by the full length coding sequence of the cDNA deposited with any ATCC accession number shown in Table 7

Isolated polypeptide comprising a.

<13th aspect>

A chimeric polypeptide comprising the polypeptide of claim 11 fused to a heterologous polypeptide.

<14th aspect>

The chimeric polypeptide of claim 13, wherein the heterologous polypeptide is an epitope tag sequence or an Fc region of an immunoglobulin.

<Fifteenth aspect>

(a) the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10);

(b) the amino acid sequence shown in Figure 6 (SEQ ID NO: 6), Figure 7 (SEQ ID NO: 7), Figure 8 (SEQ ID NO: 8), Figure 9 (SEQ ID NO: 9), or Figure 10 (SEQ ID NO: 10) without a linked signal peptide;

(c) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10). Amino acid sequence of;

(d) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. Amino acid sequence of;

(e) an amino acid sequence encoded by the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5);

(f) an amino acid encoded by the full length coding sequence of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5). order; or

(g) the amino acid sequence encoded by the full length coding sequence of the cDNA deposited with any ATCC accession number shown in Table 7

An isolated antibody that binds to a polypeptide having an amino acid sequence identity of 80% or more.

<16th aspect>

In a fifteenth aspect,

(a) the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10);

(b) the amino acid sequence shown in Figure 6 (SEQ ID NO: 6), Figure 7 (SEQ ID NO: 7), Figure 8 (SEQ ID NO: 8), Figure 9 (SEQ ID NO: 9), or Figure 10 (SEQ ID NO: 10) without a linked signal peptide;

(c) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10). Amino acid sequence of;

(d) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. Amino acid sequence of;

(e) an amino acid sequence encoded by the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5);

(f) an amino acid encoded by the full length coding sequence of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5). order; or

(g) the amino acid sequence encoded by the full length coding sequence of the cDNA deposited with any ATCC accession number shown in Table 7

An antibody that binds to a polypeptide comprising a.

<17th aspect>

The antibody of claim 15, which is a monoclonal antibody.

<18th aspect>

The antibody of claim 15, which is an antibody fragment.

<19th aspect>

The antibody of claim 15, which is a chimeric or humanized antibody.

<20th aspect>

The antibody of claim 15 conjugated to a growth inhibitor.

<21st aspect>

The antibody of claim 15 conjugated to a cytotoxic agent.

<22th aspect>

The antibody of claim 21, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleases.

<Twenty-third aspect>

The antibody of claim 21, wherein the cytotoxic agent is a toxin.

24th aspect

The antibody of claim 23, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicins.

<25th aspect>

The antibody of claim 23, wherein the toxin is a maytansinoid.

<26th aspect>

The antibody of claim 15, which is produced in a bacterium.

<27th aspect>

The antibody of claim 15, which is produced in CHO cells.

<28th aspect>

The antibody of claim 15, wherein the antibody induces death of the bound cells.

<29th aspect>

The antibody of claim 15, which is detectably labeled.

<30th aspect>

An isolated nucleic acid comprising the nucleotide sequence encoding the antibody of embodiment 15.

<31st aspect>

An expression vector comprising the nucleic acid of the thirtieth aspect, operably linked to a regulatory sequence recognized by a host cell to be transformed.

<32th aspect>

A host cell comprising the expression vector of embodiment 31.

<33th aspect>

The method of claim 32, wherein the CHO cell is E. coli. Host cells that are E. coli cells or yeast cells.

<34th aspect>

Culturing the host cell of embodiment 32 under conditions suitable for expression of the antibody, and recovering the antibody from the cell culture.

<35th aspect>

(a) the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10);

(b) the amino acid sequence shown in Figure 6 (SEQ ID NO: 6), Figure 7 (SEQ ID NO: 7), Figure 8 (SEQ ID NO: 8), Figure 9 (SEQ ID NO: 9), or Figure 10 (SEQ ID NO: 10) without a linked signal peptide;

(c) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10). Amino acid sequence of;

(d) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. Amino acid sequence of;

(e) an amino acid sequence encoded by the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5);

(f) an amino acid encoded by the full length coding sequence of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5). order; or

(g) the amino acid sequence encoded by the full length coding sequence of the cDNA deposited with any ATCC accession number shown in Table 7

An isolated oligopeptide that binds to a polypeptide having an amino acid sequence identity of 80% or more.

<36th aspect>

The method of claim 35, wherein

(a) the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10);

(b) the amino acid sequence shown in Figure 6 (SEQ ID NO: 6), Figure 7 (SEQ ID NO: 7), Figure 8 (SEQ ID NO: 8), Figure 9 (SEQ ID NO: 9), or Figure 10 (SEQ ID NO: 10) without a linked signal peptide;

(c) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10). Amino acid sequence of;

(d) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. Amino acid sequence of;

(e) an amino acid sequence encoded by the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5);

(f) an amino acid encoded by the full length coding sequence of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5). order; or

(g) the amino acid sequence encoded by the full length coding sequence of the cDNA deposited with any ATCC accession number shown in Table 7

Oligopeptides that bind to a polypeptide comprising a.

<37th aspect>

The oligopeptide of claim 35 conjugated to a growth inhibitor.

<38th aspect>

The oligopeptide of claim 35 conjugated to a cytotoxic agent.

<39th aspect>

The oligopeptide of claim 38, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleases.

<40th aspect>

The oligopeptide of claim 38, wherein the cytotoxic agent is a toxin.

<41th aspect>

The oligopeptide of claim 40, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicins.

<42th aspect>

The oligopeptide of claim 40 wherein the toxin is a maytansinoid.

<43th aspect>

The oligopeptide of claim 35, which induces death of the bound cells.

<44th aspect>

The oligopeptide of claim 35, which is detectably labeled.

<45th aspect>

(a) the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10);

(b) the amino acid sequence shown in Figure 6 (SEQ ID NO: 6), Figure 7 (SEQ ID NO: 7), Figure 8 (SEQ ID NO: 8), Figure 9 (SEQ ID NO: 9), or Figure 10 (SEQ ID NO: 10) without a linked signal peptide;

(c) the extracellular domain of the polypeptide shown in Figure 6 (SEQ ID NO: 6), Figure 7 (SEQ ID NO: 7), Figure 8 (SEQ ID NO: 8), Figure 9 (SEQ ID NO: 9), or Figure 10 (SEQ ID NO: 10) Amino acid sequence of;

(d) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. Amino acid sequence of;

(e) an amino acid sequence encoded by the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5);

(f) an amino acid encoded by the full length coding sequence of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5). order; or

(g) the amino acid sequence encoded by the full length coding sequence of the cDNA deposited with any ATCC accession number shown in Table 7

A TAT binding organic molecule that binds to a polypeptide having an amino acid sequence identity of 80% or more.

<46th aspect>

The method of embodiment 45,

(a) the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10);

(b) the amino acid sequence shown in Figure 6 (SEQ ID NO: 6), Figure 7 (SEQ ID NO: 7), Figure 8 (SEQ ID NO: 8), Figure 9 (SEQ ID NO: 9), or Figure 10 (SEQ ID NO: 10) without a linked signal peptide;

(c) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10). Amino acid sequence of;

(d) the extracellular domain of the polypeptide shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. Amino acid sequence of;

(e) an amino acid sequence encoded by the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5);

(f) an amino acid encoded by the full length coding sequence of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5). order; or

(g) the amino acid sequence encoded by the full length coding sequence of the cDNA deposited with any ATCC accession number shown in Table 7

An organic molecule that binds to a polypeptide comprising a.

<47th aspect>

The organic molecule of claim 45 conjugated to a growth inhibitor.

<48th aspect>

The organic molecule of claim 45 conjugated to a cytotoxic agent.

<49th aspect>

The organic molecule of claim 48, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes, and nucleic acid degrading enzymes.

<50th aspect>

The organic molecule of claim 48, wherein the cytotoxic agent is a toxin.

<51th aspect>

The organic molecule of claim 50, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicins.

<52th aspect>

The organic molecule of claim 50, wherein the toxin is a maytansinoid.

<53th aspect>

The organic molecule of claim 45, which induces death of the bound cells.

<54th aspect>

The organic molecule of claim 45, which is detectably labeled.

<Fifth aspect>

(a) the polypeptide of embodiment 11; (b) the chimeric polypeptide of embodiment 13; (c) the antibody of embodiment 15; (d) the oligopeptide of embodiment 35; Or (e) a TAT binding organic molecule of embodiment 45 and a carrier.

56th Embodiment

The composition of claim 55 wherein the carrier is a pharmaceutically acceptable carrier.

<57th Embodiment>

(a) a container; And (b) the composition of the 55th aspect contained in said container

Manufacturing article comprising a.

58th Embodiment

The article of manufacture of claim 57, further comprising a label attached to the container or a package insert contained within the container indicating that the composition is used for therapeutic treatment or diagnostic detection of cancer.

<59th aspect>

(a) the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10); Or (b) encoded by a nucleotide sequence comprising the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. Contacting a cancer cell expressing a polypeptide having an amino acid sequence identity of at least 80% with an amino acid sequence of the amino acid sequence and an antibody, oligopeptide, or organic molecule binding to the polypeptide on the cancer cell, thereby killing the cancer cell, (a) FIG. 6 ( SEQ ID NO: 6), Figure 7 (SEQ ID NO: 7), Figure 8 (SEQ ID NO: 8), Figure 9 (SEQ ID NO: 9), or Figure 10 (SEQ ID NO: 10); Or (b) encoded by a nucleotide sequence comprising the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. A method for killing cancer cells expressing a polypeptide having an amino acid sequence identity of 80% or more with an amino acid sequence.

 <60th aspect>

The method of claim 59, wherein the antibody is a monoclonal antibody.

<61th aspect>

The method of claim 59, wherein the antibody is an antibody fragment.

<62th aspect>

The method of claim 59, wherein the antibody is a chimeric or humanized antibody.

<63th Embodiment>

60. The method of claim 59, wherein the antibody, oligopeptide or organic molecule is conjugated to a growth inhibitor.

<64th Embodiment>

The method of claim 59, wherein the antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.

<65th Embodiment>

The method of claim 64, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleases.

<66th Embodiment>

The method of claim 64, wherein the cytotoxic agent is a toxin.

<67th aspect>

The method of claim 66, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicins.

<68th aspect>

The method of claim 66, wherein the toxin is a maytansinoid.

<69th aspect>

The method of claim 59, wherein the antibody is produced in bacteria,

<70th aspect>

The method of claim 59, wherein the antibody is produced in CHO cells,

<71th aspect>

60. The method of claim 59, wherein the cancer cells are further exposed to radiation therapy or chemotherapeutic agents.

<72th aspect>

The cancer cell of claim 59, wherein the cancer cell is selected from the group consisting of breast cancer cells, colorectal cancer cells, lung cancer cells, ovarian cancer cells, central nervous system cancer cells, liver cancer cells, bladder cancer cells, pancreatic cancer cells, cervical cancer cells, melanoma cells and leukemia cells Way.

<73th aspect>

60. The method of claim 59, wherein the cancer cells overexpress the polypeptide as compared to normal cells of the same tissue.

<74th aspect>

(a) the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10); Or (b) encoded by a nucleotide sequence comprising the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5). Effectively treating the mammal by administering a therapeutically effective amount of an antibody, oligopeptide, or organic molecule that binds the polypeptide to a mammal having a tumor comprising a cell expressing a polypeptide having at least 80% amino acid sequence identity with the resulting amino acid sequence. (A) the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10); Or (b) encoded by the nucleotide sequence comprising the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. A method of therapeutically treating a mammal with a tumor comprising a cell expressing a polypeptide having at least 80% amino acid sequence identity with an amino acid sequence.

<75th aspect>

The method of claim 74, wherein the antibody is a monoclonal antibody.

<76th Embodiment>

The method of claim 74, wherein the antibody is an antibody fragment.

<77th aspect>

The method of claim 74, wherein the antibody is a chimeric or humanized antibody.

<78th Aspect>

The method of claim 74, wherein the antibody, oligopeptide or organic molecule is conjugated to a growth inhibitor.

<79th aspect>

The method of claim 74, wherein the antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.

<80th aspect>

79. The method of claim 79, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleases.

<81th aspect>

80. The method of claim 79, wherein the cytotoxic agent is a toxin.

<82th Aspect>

The method of claim 81, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicins.

<83th aspect>

82. The method of claim 81, wherein the toxin is a maytansinoid.

<84th aspect>

The method of claim 74, wherein the antibody is produced in bacteria.

<85th aspect>

The method of claim 74, wherein the antibody is produced in CHO cells.

<86th aspect>

The method of claim 74, wherein the cancer cells are further exposed to radiation therapy or chemotherapeutic agents.

<87th aspect>

The method of claim 74, wherein the tumor is a breast tumor, colorectal tumor, lung tumor, ovarian tumor, central nervous system tumor, liver tumor, bladder tumor, pancreatic tumor or neck tumor.

<88th aspect>

(a) the amino acid sequence shown in FIG. 6 (SEQ ID NO: 6), FIG. 7 (SEQ ID NO: 7), FIG. 8 (SEQ ID NO: 8), FIG. 9 (SEQ ID NO: 9), or FIG. 10 (SEQ ID NO: 10); Or (b) encoded by a nucleotide sequence comprising the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5). Exposing a sample suspected of containing a polypeptide having at least 80% amino acid sequence identity with an amino acid sequence to be exposed to an antibody, oligopeptide or organic molecule that binds to the polypeptide, and the polypeptide and the antibody, oligopeptide or organic in the sample (A) shown in Figure 6 (SEQ ID NO: 6), Figure 7 (SEQ ID NO: 7), Figure 8 (SEQ ID NO: 8), Figure 9 (SEQ ID NO: 9), or Figure 10 (SEQ ID NO: 10), comprising measuring the binding of a molecule Amino acid sequence; Or (b) encoded by a nucleotide sequence comprising the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5). A method for determining the presence of a polypeptide in a sample suspected of containing a polypeptide having at least 80% amino acid sequence identity with an amino acid sequence.

<89th aspect>

89. The method of claim 88, wherein the sample comprises cells suspected of expressing said polypeptide.

<90th aspect>

90. The method of claim 89, wherein the cell is a cancer cell.

<91th aspect>

89. The method of claim 88, wherein the antibody, oligopeptide or organic molecule is detectably labeled.

<92th aspect>

(A) Figure 6 (SEQ ID NO: 6), Figure 7 (SEQ ID NO: 7), Figure 8 (SEQ ID NO: 8), Figure 9 in a test sample of tissue cells obtained from a mammal and a control sample derived from the same tissue of known normal cells. (SEQ ID NO: 9) or the amino acid sequence shown in Fig. 10 (SEQ ID NO: 10); Or (b) encoded by a nucleotide sequence comprising the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5). Detecting an expression level of a gene encoding a polypeptide having an amino acid sequence identity of 80% or more with an amino acid sequence, wherein the expression level of the polypeptide in the test sample is higher than that of the control sample. A method for diagnosing the presence of a tumor in a mammal, said method being indicative of the presence of a tumor in the obtained mammal.

<93th aspect>

92. The method of claim 92, wherein detecting the expression level of the gene encoding the polypeptide comprises in situ hybridization or RT-PCR analysis using the oligonucleotide.

<94th aspect>

93. The method of claim 92, wherein detecting the expression level of a gene encoding a polypeptide comprises immunohistochemical analysis using the antibody.

<95th aspect>

Test samples of tissue cells obtained from a mammal are shown in (a) Figure 6 (SEQ ID NO: 6), Figure 7 (SEQ ID NO: 7), Figure 8 (SEQ ID NO: 8), Figure 9 (SEQ ID NO: 9), or Figure 10 (SEQ ID NO: 10). Amino acid sequence; Or (b) encoded by a nucleotide sequence comprising the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 2), FIG. 3 (SEQ ID NO: 3), FIG. 4 (SEQ ID NO: 4), or FIG. 5 (SEQ ID NO: 5). Contacting an antibody, oligopeptide or organic molecule that binds to a polypeptide having an amino acid sequence identity of at least 80% with the resulting amino acid sequence, and detecting the formation of a complex between the antibody, oligopeptide or organic molecule and the polypeptide in a test sample. And wherein the complex formation is indicative of the presence of a tumor in said mammal.

<96th aspect>

95. The method of claim 95, wherein the antibody, oligopeptide or organic molecule is detectably labeled.

<97th aspect>

The method of claim 95, wherein the test sample of tissue cells is from an individual suspected of having a cancerous tumor.

Additional embodiments of the invention will be apparent to those of ordinary skill in the art upon reading this specification.

I. Definition

As used herein, the terms “TAT polypeptide” and “TAT” refer to a variety of polypeptides, followed immediately by numbers, with the full name (ie, TAT / number) refers to a specific polypeptide sequence as described herein. The terms "number" in the terms "TAT / number polypeptide" and "TAT / number" are represented by actual numbers as used herein, including native sequence polypeptides, polypeptide variants, and fragments of native sequence polypeptides and polypeptide variants ( Further defined herein). TAT polypeptides described herein can be isolated from various sources, such as human tissue or other sources, or can be prepared by recombinant or synthetic methods. The term "TAT polypeptide" refers to each individual TAT / number polypeptide disclosed herein. All disclosures referring to "TAT polypeptides" herein refer to each of these polypeptides individually as well as collectively. For example, preparation of TAT polypeptide, purification thereof, induction thereof, formation of antibodies thereto, formation of TAT binding oligopeptides thereto, formation of TAT binding organic molecules thereto, administration thereof, compositions containing the same, methods of treating diseases using the same And the like correspond to each individual polypeptide of the present invention. The term "TAT polypeptide" also includes variants of the TAT / number polypeptides disclosed herein.

"Native sequence TAT polypeptide" includes polypeptides having the same amino acid sequence as the corresponding TAT polypeptide derived from nature. Such native sequence TAT polypeptides may be isolated from nature or may be prepared by recombinant or synthetic means. The term “natural sequence TAT polypeptide” specifically refers to a naturally occurring truncated or secreted form (eg, extracellular domain sequence) of a particular TAT polypeptide, a naturally occurring variant form of the polypeptide (eg, And in a naturally occurring allelic variant of said polypeptide. In certain embodiments of the invention, the native sequence TAT polypeptide disclosed herein is a mature or full length native sequence polypeptide comprising the full length amino acid sequence shown in the accompanying drawings. Start and stop codons are indicated in bold and underlined in the figures (if indicated). Nucleic acid residues represented by "N" in the accompanying drawings are any nucleic acid residues. However, although the TAT polypeptides disclosed in the accompanying drawings are shown to begin with methionine residues designated at amino acid position 1 in the figures, other methionine residues located upstream or downstream from amino acid position 1 in the figures may be used as starting amino acid residues of the TAT polypeptide. It can be conceived and also possible.

TAT polypeptide "extradomain" or "ECD" refers to the form of said TAT polypeptide that is essentially free of transmembrane and cytoplasmic domains. Typically, the TAT polypeptide ECD will contain less than 1% of such transmembrane and / or cytoplasmic domains, preferably less than 0.5% of these domains. It will be appreciated that any transmembrane domain identified in a TAT polypeptide of the invention has been identified according to criteria commonly used to identify hydrophobic domain types in the art. The exact boundaries of the transmembrane domain may vary, but most will vary at about 5 amino acids or less at either end of this domain, as initially identified herein. Thus, the extracellular domain of the TAT polypeptide may optionally contain up to about 5 amino acids at either interface of the transmembrane domain / extracellular domain identified in the Examples or the description, with or without a linked signal peptide. Such polypeptides and nucleic acids encoding them are contemplated herein.

Approximate positions of "signal peptides" of the various TAT polypeptides disclosed herein are shown herein and / or in the accompanying drawings. However, it should be noted that although the C-terminal boundary of the signal peptide may vary, most of the amino acids may vary in about 5 or less at either interface of the signal peptide C-terminal first identified herein, where the signal The C-terminal boundary of a peptide can be identified according to the criteria commonly used to identify the type of amino acid sequence element in the art (see, eg, Nielsen et al., Prot. Eng. 10: 1-6 ( 1997) and von Heinje et al., Nucl.Acids. Res. 14: 4683-4690 (1986). In addition, it should be recognized that in some cases cleavage of the signal sequence from the secreting polypeptide is not uniform throughout, resulting in one or more secreted polypeptides. These mature polypeptides and polynucleotides encoding them in which the signal peptide is cleaved within the range of about 5 or less amino acids on either interface of the C-terminus of the signal peptide identified herein are contemplated herein.

A "TAT polypeptide variant" refers to a full length native sequence TAT polypeptide sequence as described herein, a TAT polypeptide sequence without a signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide with or without a signal peptide as described herein or As defined herein, the amino acid sequence identity with any other fragment of the full length TAT polypeptide sequence as disclosed (eg, a fragment encoded by a nucleic acid encoding only a portion of the full coding sequence of the full length TAT polypeptide) is at least about 80%. TAT polypeptide as described herein, preferably active TAT polypeptide. Examples of such TAT polypeptide variants include TAT polypeptides having one or more amino acid residues added or deleted at the N-terminus or C-terminus of the full-length natural amino acid sequence. Typically, the TAT polypeptide variant is a full-length native sequence TAT polypeptide sequence as disclosed herein, a TAT polypeptide sequence without a signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide with or without a signal peptide as described herein or Amino acid sequence identity with any other specially defined fragment of the full-length TAT polypeptide sequence as disclosed in at least about 80%, alternatively about 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more. Typically, the length of the TAT variant polypeptide is about 10 or more amino acids, alternatively about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 , 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 , 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, There are 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 and more. Optionally, the TAT variant polypeptide may be conservatively substituted only one amino acid relative to the native TAT polypeptide sequence, or only two, three, four, five, six, seven, Only 8, 9 or 10 amino acids have been conservatively substituted.

With respect to the TAT polypeptide sequence identified herein, “% amino acid sequence identity” refers to aligning the sequence and, if necessary, introducing any conservative substitutions after introducing a gap to obtain the maximum sequence identity. Defined as a percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular TAT polypeptide sequence without consideration as part of sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished using a variety of methods that belong to the art, such as computer software that is publicly available, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Can be achieved. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for purposes herein,% amino acid sequence identity values are obtained using the sequence comparison computer program ALIGN-2, the complete source code of the ALIGN-2 program is described in Table 1 below. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and the source code shown in Table 1 below is stored as a user document in the U.S. Copyright Office (Washington, DC 20559), U.S. Copyright Registration TXU510087 Registered as The ALIGN-2 program is Genentech, Inc. (South San Francisco, Calif., USA) is publicly available or can be compiled from the source code listed in Table 1. The ALIGN-2 program can be compiled and used on a UNIX working system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

When ALIGN-2 is used for amino acid sequence comparison, a given amino acid sequence B for a given amino acid sequence B, B, or a given amino acid sequence A for B (also, a given amino acid sequence identity for a given amino acid sequence B, B, or B) % Amino acid sequence identity of a given amino acid sequence A having or including (%) may be expressed as follows:

X / Y × 100

Where X is the number of amino acid residues recorded as equally matched by the sequence alignment program ALIGN-2 in the program alignment of A and B, and Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity of A to B will not be equal to the amino acid sequence identity of B to A to A. As an example of calculating the amino acid sequence identity (%) using this method, Table 2 and Table 3 calculate the amino acid sequence identity (%) of the amino acid sequence referred to as "comparative protein" to the amino acid sequence referred to as "TAT". Wherein "TAT" refers to the amino acid sequence of the TAT polypeptide of the family of interest, "comparative protein" refers to the amino acid sequence of the polypeptide to be compared with the "TAT" polypeptide of interest, and "X", "Y". And "Z" each represent amino acid residues of different assumptions. Unless otherwise specified, all percent amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

A "TAT variant polynucleotide" or "TAT variant nucleic acid sequence" is a nucleic acid molecule encoding a TAT polypeptide, preferably an active TAT polypeptide, as defined herein, the full length native sequence TAT polypeptide sequence as disclosed herein, Full length native sequence TAT polypeptide sequence without signal peptide as disclosed herein, extracellular domain of TAT polypeptide with or without signal peptide as disclosed herein, or any other fragment (eg, full length TAT polypeptide sequence as disclosed herein) Nucleic acid sequence identity with a nucleic acid sequence encoding a fragment encoded by a nucleic acid encoding only a portion of the full coding sequence of the full length TAT polypeptide) is at least about 80%. Typically, the TAT variant polynucleotides comprise a full length native sequence TAT polypeptide sequence as described herein, a full length native sequence TAT polypeptide sequence without signal peptide as described herein, or a TAT polypeptide with or without signal peptide as described herein. Nucleic acid sequence identity with the extracellular domain or nucleic acid sequence encoding any other fragment of the full-length TAT polypeptide sequence as disclosed herein is at least about 80%, alternatively about 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more. Variants do not include native nucleotide sequences.

Typically, the length of a TAT variant polynucleotide is at least about 5 nucleotides, alternatively about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125 , 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220 , 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550 , 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720 , 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930 , 940, 950, 960, 970, 980, 990 or 1000 or more, wherein the term "about" in this context means the nucleotide sequence length ± 10% of this length.

With respect to the TAT-encoding nucleic acid sequences identified herein, “% nucleic acid sequence identity” refers to nucleotides of the TAT nucleic acid sequence of interest after introducing a gap to align the sequences and, if necessary, to obtain the maximum sequence identity (%). It is defined as a percentage of nucleotides of the same candidate sequence. Alignment to determine percent nucleic acid sequence identity can be accomplished using various methods within the art, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalin (DNASTAR) software. have. However, for purposes herein,% nucleic acid sequence identity values are obtained using the sequence comparison computer program ALIGN-2, the complete source code of the ALIGN-2 program is listed in Table 1 below. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc. The source code shown in Table 1 is stored as a user document in the U.S. Copyright Office (Washington, DC 20559), and registered under U.S. Copyright Registration TXU510087. have. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, CA, or may be compiled from the source code listed in Table 1. The ALIGN-2 program can be compiled and used on a UNIX working system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

When ALIGN-2 is used for nucleic acid sequence comparison, given nucleic acid sequence D, with D, or with given nucleic acid sequence C for D (with given nucleic acid sequence D, with D, or with constant nucleic acid sequence identity to D, % Nucleic acid sequence identity of a given nucleic acid sequence C having or including) is calculated as follows:

W / Z × 100

Where W is the number of nucleotides recorded as equally matched by the sequence alignment program ALIGN-2 in the program alignment of C and D, and Z is the total number of nucleotides in D. It will be appreciated that if the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, then the percent nucleic acid sequence identity of C to D is not equal to the percent nucleic acid sequence identity of D to C. Examples of nucleic acid sequence identity calculations, for example, Tables 4 and 5 show methods of calculating nucleic acid sequence identity (%) of nucleic acid sequences referred to as "comparative DNA" to nucleic acid sequences referred to as "TAT-DNA". "TAT-DNA" indicates the TAT-encoding nucleic acid sequence of the hypothesis of interest, "comparative DNA" indicates the nucleotide sequence of the nucleic acid molecule to be compared with the "TAT-DNA" nucleic acid molecule of interest, "N", "L" And "V" each represent a different assumption of nucleotides. Unless otherwise stated, all percent nucleic acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

In other embodiments, the TAT variant polynucleotide is a nucleic acid molecule encoding a TAT polypeptide, and may be hybridized to a nucleotide sequence encoding a full length TAT polypeptide as disclosed herein under stringent hybridization conditions and wash conditions. The TAT variant polypeptide may be one encoded by a TAT variant polynucleotide.

When “isolating” is used to describe the various TAT polypeptides disclosed herein, it is meant polypeptides that have been identified and separated and / or recovered from natural environmental components. Typically, the contaminating component of the polypeptide's natural environment is a substance that prevents the polypeptide from being used for diagnosis or treatment, examples of which include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the polypeptide is (1) to a degree sufficient to obtain an N-terminal or internal amino acid sequence consisting of 15 or more residues when using a rotating cup sequencer, or (2) Coomassie Blue Or preferably using silver staining to the extent that only one band appears upon SDS-PAGE under reducing or non-reducing conditions. Isolated antibody includes the polypeptide in situ within recombinant cells since at least one TAT polypeptide natural environmental component will not be present. Ordinarily, however, isolated polypeptide will be prepared via one or more purification steps.

An “isolated” TAT polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule identified and isolated from one or more contaminating nucleic acid molecules that are typically bound in a natural source of said polypeptide-encoding nucleic acid. Isolated polypeptide-encoding nucleic acid molecules exist differently from the forms or states found in nature. Thus, isolated polypeptide-encoding nucleic acid molecules are distinguished from certain polypeptide-encoding nucleic acid molecules present in natural cells. However, isolated polypeptide-encoding nucleic acid molecules include, for example, polypeptide-encoding nucleic acid molecules that are at different chromosomal positions than in natural cells and are typically contained in cells expressing the polypeptide.

The term "regulatory sequence" refers to a DNA sequence necessary for the expression of a coding sequence operably linked in a particular host organism. Examples of regulatory sequences suitable for prokaryotes include promoters, optionally operator sequences and ribosomal binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic acids are “operably linked” when placed in a functional relationship with other nucleic acid sequences. For example, the DNA of a presequence or secretion leader is operably linked to the DNA of the polypeptide when the polypeptide is expressed as a preprotein involved in its secretion, and the promoter or enhancer When affecting the transcription of the coding sequence of the polypeptide of interest, the coding sequence is operably linked, and the ribosome binding site is operably linked to the coding sequence when placed to facilitate translation of the coding sequence. Typically, “operably linked” means that the DNA sequences to be linked are located contiguously, and in the case of a secretory leader, not only are located contiguous but also within the same reading frame. However, enhancers do not have to be contiguous. Linking is accomplished through ligation at convenient restriction enzyme sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

The "stringency" of the hybridization reaction can be readily determined by one skilled in the art and is an experimental calculation that typically depends on probe length, wash temperature and salt concentration. Typically, the longer the length of the probe, the higher the temperature required for proper annealing; The shorter the probe, the lower the temperature required. Typically, hybridization depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Thus, the higher the relative temperature, the more stringent the reaction conditions, while the lower the relative temperature, the less stringent the reaction conditions. For more detailed information and explanations regarding the stringency of the hybridization reaction, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995).

"Strict conditions" or "high stringency conditions" as defined herein means (1) 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dode at conditions of low ionic strength and high temperature at washing, for example at 50 ° C. Conditions using silsulfate, (2) 0.1% bovine serum albumin / 0.1% picol / 0.1% polyvinylpyrroli containing formamide, e.g., 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C upon hybridization Conditions using a denaturant such as 50% (volume / volume) formamide with DON / 50 mM sodium phosphate buffer (pH 6.5), or (3) 50% formamide at 42 ° C., 5 × SSC (0.75 M NaCl , 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS and 10% Dextran sulfate was used and 0.2 × SSC (sodium chloride / sodium citrate) at 42 ° C. After washing with 50% formamide at 55 ° C., and performing a strict washing with 0.1 × SSC containing EDTA at 55 ° C.

"Medium stringency conditions" can be found as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press 1989, and are less stringent washing solutions and hybridization conditions than those described above. (E.g., percentage of temperature, ionic strength and SDS). Examples of moderate stringency conditions include 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate and The solution is incubated at 37 ° C. overnight in a solution containing 20 mg / ml of salmon sperm truncated denatured DNA, followed by washing the filter with 1 × SSC at about 37-50 ° C. Those skilled in the art will be well aware of how to adjust the required temperature, ionic strength and the like in accordance with factors such as probe length and the like.

As used herein, the term “epitope tagged” refers to a chimeric polypeptide comprising a TAT polypeptide or anti-TAT antibody fused to a “tag polypeptide”. The tag polypeptide has enough residues to provide enough epitope for the antibody to be made but short enough to not interfere with the activity of the TAT polypeptide to be fused. It is also desirable that the tag polypeptide be very unique so that the antibody against itself does not substantially cross react with other epitopes. Typically, the amino acid residues of suitable tag polypeptides are at least 6, usually about 8 to 50 (preferably about 10 to 20).

For the purposes herein, "active" or "active" refers to the form (s) of a TAT polypeptide that retains the biological and / or immunological activity of a naturally occurring or naturally occurring TAT polypeptide, wherein the "biological" activity is natural or Not the ability to induce antibody production against antigenic epitopes in naturally occurring TATs, but refers to biological functions (inhibitory or stimulatory functions) by naturally or naturally occurring TATs, and "immunological" activity refers to naturally occurring or naturally occurring TATs. Refers to the ability to induce antibody production against antigenic epitopes.

The term “antagonist” is used in its broadest sense and refers to any molecule that partially or completely blocks, inhibits or neutralizes the biological activity of the native TAT polypeptides disclosed herein. In a similar manner, the term "agonist" is used in its broadest sense and refers to any molecule that mimics the biological activity of the native TAT polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include antibodies or antibody fragments of agonist or antagonists, fragments or amino acid sequence variants of native TAT polypeptide, peptides, antisense oligonucleotides, organic small molecules and the like. Methods for identifying an agonist or antagonist of a TAT polypeptide include contacting the TAT polypeptide with a candidate agonist molecule or a candidate antagonist molecule, and determining a detectable change in one or more biological activities normally associated with the TAT polypeptide. can do.

"Treating", "treatment" or "relaxation" refers to both therapeutic treatment and prophylactic or preventative treatment, which aims to prevent or alleviate (reduce) targeted pathological symptoms or diseases. Subjects in need of treatment include those already afflicted with the disease, as well as subjects susceptible to the disease or subjects to which diseases are to be prevented. Reduction in the number of cancer cells or absence of cancer cells after administration of a therapeutically effective amount of an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule in accordance with the methods of the invention; Reduction in tumor size; Inhibition (ie, slowing to some extent and preferably stopping) of cancer cell infiltration into peripheral organs, including the spread of cancer to soft tissues and bones; Inhibit (ie, slow to some extent and preferably stop) tumor metastasis; Inhibition of tumor growth (to some extent); And / or relieve (to some extent) one or more symptoms associated with the particular cancer; If one or more of reducing mortality and morbidity and improving quality of life have not been reduced or shown to the extent observable and / or measurable in the patient, the subject or mammal has been successfully "treated" for TAT polypeptide-expressing cancer. will be. If the anti-TAT antibody or TAT binding oligopeptide is capable of disrupting the growth of existing cancer cells and / or killing existing cancer cells, this may indicate cell arrest and / or cytotoxicity. Reduction of these signs or symptoms may be felt by the patient.

The parameters for evaluating successful treatment and improvement of disease can be readily determined by conventional methods known to the physician. For cancer therapy, treatment efficacy can be measured, for example, by evaluating the time spent on disease progression (TTP) and / or determining the response rate (RR). Metastasis can be identified by staging tests and bone scans and tests by measuring calcium levels and other enzymes to determine if cancer has spread to bone. A CT scan can also be done to see if cancer has spread to the pelvis and lymph nodes. Chest X-rays and measurements of liver enzyme levels by known methods are used to confirm metastasis to the lung and liver, respectively. Other conventional methods for monitoring the disease include transrectal ultrasonography (TRUS) and transrectal needle biopsy (TRNB).

In the case of bladder cancer, a more limited form of cancer, urological examination of the urinary cell by cystoscopy, monitoring of the presence of blood in the urine, observation of the urinary tract epithelium by sonic holography or intravenous pyelogram, and computer Tomography (CT) and magnetic resonance imaging (MRI). The presence of distant metastasis can be investigated by abdominal CT, chest X-rays or radionuclide imaging of the skeleton.

"Chronic" administration refers to administering the agent (s) in a continuous manner as opposed to an acute mode so that the initial therapeutic effect (activity) is maintained for an extended period of time. "Intermittent" administration is a treatment characterized by periodic, rather than continuous, interruption.

“Mammals” for treating or alleviating the symptoms of cancer include humans, livestock and animal husbandry animals and zoos, sports or pets such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. It refers to any animal classified as a mammal, including. Preferably, the mammal is a human.

Administration in combination with one or more other therapeutic agents includes simultaneous (co) administration and continuous administration in any order.

As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilization that is nontoxic to a cell or mammal exposed to it at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffer solution. Examples of physiologically acceptable carriers include buffers such as phosphoric acid, citric acid and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt-forming counterions such as sodium; And / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG) and PLURONICS®.

"Solid phase" or "solid support" means a non-aqueous matrix to which an antibody, TAT binding oligopeptide or TAT binding organic molecule of the invention can be attached or attached. Examples of solid phases encompassed herein include solids, polysaccharides (eg agarose), polyacrylamides, polystyrenes, polyvinyl alcohols and silicones formed partially or completely from glass (eg pore controlled glass). . In certain embodiments, depending on the content, the solid phase may comprise the wells of an analytical plate, and in other embodiments the solid phase is a preparative column (eg, an affinity chromatography column). The term also encompasses discrete particles in discrete particles, such as those described in US Pat. No. 4,275,149.

A "liposome" is a small vesicle composed of various forms of lipids, phospholipids, and / or surfactants useful for delivering a drug (eg, a TAT polypeptide or antibody or TAT binding oligopeptide) to a mammal. Typically, the components of the liposomes are aligned in bilayer form, similar to the lipid alignment of the biofilm.

"Small molecule" or organic "small molecule" is defined herein as having a molecular weight of less than about 500 Daltons.

An “effective amount” of a polypeptide, antibody, TAT binding oligopeptide, TAT binding organic molecule or agonist or antagonist thereof as disclosed herein is an amount sufficient to accomplish the specifically stated purpose. In relation to the stated purpose, an "effective amount" can be determined empirically and in a customary manner.

The term "therapeutically effective amount" refers to an amount of an antibody, polypeptide, TAT binding oligopeptide, TAT binding organic molecule or other drug that is effective for "treatment" of a disease or condition in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug may be a decrease in the number of cancer cells; Reduction in tumor size; Inhibit (ie, slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; Inhibit (ie, slow to some extent and preferably stop) tumor metastasis; Inhibition of tumor growth (to some extent); And / or relieve (to some extent) one or more symptoms associated with the cancer. See the definition of "treatment". As long as it can interfere with the growth of existing cancer cells and / or kill existing cancer cells, this may indicate cell arrest and / or cytotoxicity.

The "growth inhibition" of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule is an amount capable of inhibiting the growth of cells, in particular tumors, eg cancer cells, in vitro or in vivo. The "growth inhibition" of anti-TAT antibodies, TAT polypeptides, TAT binding oligopeptides or TAT binding organic molecules for inhibiting neoplastic cell growth can be determined empirically and in a customary manner.

The “cytotoxic amount” of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule is an amount that can cause the destruction of cells, in particular tumors, eg cancer cells, in vitro or in vivo. The "cytotoxic amount" of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule to inhibit neoplastic cell growth can be determined empirically and in a customary manner.

The term “antibody” is used in its broadest sense and specifically refers to, for example, single anti-TAT monoclonal antibodies (including agonists, antagonists and neutralizing antibodies), anti-TAT antibody compositions with polyepithos specificity, poly Clonal antibodies, single-chain anti-TAT antibodies and fragments of anti-TAT antibodies (see below), provided they exhibit the desired biological or immunological activity. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

An "isolated antibody" is an antibody that has been identified and separated and / or recovered from components of its natural environment. Contaminant components of the natural environment are substances that prevent antibodies from being used for diagnosis or treatment, and examples thereof include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody comprises (1) greater than 95% by weight of the antibody, most preferably greater than 99% by weight, as measured by the Lowry method, and (2) 15 using a rotating cup sequencer. To an extent sufficient to obtain an N-terminal or internal amino acid sequence consisting of two or more residues, or (3) one can be subjected to SDS-PAGE under reducing or non-reducing conditions using Coomassie Blue or preferably silver staining. Purify enough to show only bands. Isolated antibody includes the antibody in situ within recombinant cells, since at least one antibody natural environment component will not be present. Ordinarily, isolated antibodies will be prepared through one or more purification steps.

The basic four-chain antibody unit is a heterotetramer glycoprotein consisting of two identical light chains (L) and two identical heavy chains (H) (IgM antibodies have five basic heterotetramer units with an additional polypeptide called the J chain). It contains 10 antigen binding sites, but secreted IgA antibody can be polymerized to form a multivalent assembly comprising 2 to 5 basic four-chain units together with the J chain). For IgG, the four-chain unit is approximately about 150,000 Daltons. Each L chain is linked to the H chain by one covalent disulfide bond, but the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. In addition, there are also intrachain disulfide bridges that are spaced apart at regular intervals in each H chain and L chain. At the N-terminus of each H chain is the variable domain (V _H ), followed by three constant domains (C _H ) for each of the α and γ chains, and four for the μ and ε isotypes. There is a C _H domain. There is a variable domain (V _L ) at the N-terminus of each L chain and a constant domain (C _L ) at the opposite end. V _L is aligned with V _H and C _L is aligned with the first constant domain (C _H 1) of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. Pairing of V _H and V _L together forms a single antigen-binding site. For structures and properties of antibodies belonging to different classes, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT. , 1994, page 71 and Chapter 6].

The L chains of any vertebrate species can be one of two distinctly different types called kappa and lambda, depending on the amino acid sequence of their constant domains. Immunoglobulins can be classified into various classes or isotypes depending on the amino acid sequence of the constant domain of the heavy chain (C _H ). There are five classes of immunoglobulins, namely IgA, IgD, IgE, IgG and IgM with heavy chains called α, δ, ε, γ and μ, respectively. The γ and α classes are further classified into subclasses based on relatively small differences in C _H sequence and function, for example humans express subclasses IgGl, IgG2, IgG3, IgG4, IgA1 and IgA2.

The term “variable” refers to the fact that certain fragments of the variable domains exhibit a wide range of sequence differences between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for a particular antigen. However, this variability is not evenly distributed throughout the 110 amino acids of the variable domain. Instead, the V region is a framework region consisting of 15 to 30 amino acids, 9 to 12 amino acids in length and separated by a shorter region called the "hypervariable region" with extremely high variability. It consists of a relatively constant stretch called). The variable domains of each of the natural heavy and light chains predominantly comprise a β-sheet structure and comprise four FRs linked by three hypervariable regions, which FR connect the β-sheet structure, and in some cases the A loop forms to form part of the β-sheet structure. The hypervariable regions in each chain are located close to each other by the FRs, and the hypervariable regions from the other chain contribute to the formation of the antigen-binding site of the antibody (Kabat et al., Sequences of Proteins of Immunological Interest). , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). The constant domains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions such as the antibody's involvement in antibody-dependent cell-mediated cytotoxicity (ADCC).

As used herein, the term “hypervariable region” refers to an amino acid residue of an antibody that is responsible for antigen-binding. Typically, such hypervariable regions are amino acid residues from the "complementarity determining regions" or "CDRs" (eg, residues in V _L about 24 to 34 (L1), 50 to 56 (L2) and 89 to 97 (L3). ) And near residues 1 to 35 (H1), 50 to 65 (H2) and 95 to 102 (H3) in V _H (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD. (1991))) and / or amino acid residues from "hypervariable loops" (eg, residues 26-32 (L1), 50-52 (L2) and 91- to V- _L ). Residues 26-32 (H1), 53-55 (H2) and 96-101 (H3) in 96 (L3) and V _H (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)) Include.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, ie the individual antibodies that make up this population are identical except for possible naturally occurring mutations that may be present in small amounts. Do. Monoclonal antibodies are highly specific for a single antigenic site. Further, in contrast to polyclonal antibody preparations comprising different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to these specificities, monoclonal antibodies are advantageous in that they can be synthesized uncontaminated by other antibodies. The modifier "monoclonal" should not be construed to mean that antibody production is required by any particular method. For example, monoclonal antibodies useful in the present invention can be prepared by the hybridoma method first described in Kohler et al., Nature, 256: 495 (1975), or bacteria, eukaryotic animals Or recombinant DNA methods in plant cells (see, eg, US Pat. No. 4,816,567). In addition, “monoclonal antibodies” are described, for example, in Clackson et al., Nature, 352: 624-628 (1991); and Marks et al., J. Mol. Biol., 222: 581-597 (1991), can also be isolated from phage antibody libraries.

The monoclonal antibodies herein have the same chain (s), although some of the heavy and / or light chains are identical or homologous to the corresponding sequences of antibodies derived from a particular species or belonging to a particular antibody class or subclass. The remainder of includes not only antibodies derived from other species or belonging to different antibody classes or subclasses, but also “chimeric” antibodies that are identical or homologous to the corresponding sequences of the antibody fragments, provided that Activity) (see US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include “primatized” antibodies comprising variable domain antigen-binding sequences and human constant region sequences derived from non-human primates (eg, apes, apes, etc.).

An "intact" antibody is an antibody comprising an antigen binding site as well as one or more of C _L and heavy chain constant domains C _H 1, C _H 2 and C _H 3. Such constant domains may be native sequence constant domains (eg human native sequence constant domains) or amino acid sequence variants thereof. Intact antibodies preferably have one or more effector functions.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding region or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') ₂ and Fv fragments; Diabodies; Linear antibodies (see Example 2 of US Pat. No. 5,641,870, Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995)); Single-chain antibody molecules; And multispecific antibodies formed from antibody fragments.

Cleavage of the antibody with papain yields two identical antigen-binding fragments called "Fab" fragments and the remaining "Fc" fragments (this name reflects the ability to readily crystallize). The Fab fragment consists of the variable region domain of the H chain (V _H ) and the first constant domain of one heavy chain (C _H 1) and the entire L chain. Each Fab fragment has a monovalent, ie single antigen-binding site for antigen binding. Pepsin treatment of the antibody roughly corresponds to two disulfide linked Fab fragments, which exhibit bivalent antigen-binding activity, resulting in a large single F (ab ′) ₂ fragment that is still capable of cross-linking the antigen. Fab 'fragments also differ from Fab fragments by the addition of several residues at the carboxy terminus of the C _H 1 domain, including the presence of one or more cysteines from the antibody hinge region. As used herein, Fab'-SH is the name for Fab 'bearing a free thiol group in the cysteine residue (s) of the constant domains. F (ab ') ₂ antibody fragments originally were produced as pairs of Fab' fragments, with hinge cysteines between the Fab 'fragments. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chains joined together by disulfide bonds. The effector function of an antibody is determined by the sequence of the Fc region, which is also the site recognized by the Fc receptor (FcR) found in certain types of cells.

"Fv" is the minimum antibody fragment which contains a complete antigen-recognition site and an antigen-binding site. This fragment consists of a dimer in which one heavy chain variable region domain and one light chain variable region domain are tightly linked to each other by non-covalent bonds. These two domains are folded to form six hypervariable loops (three loops from the H and L chains), which provide amino acid residues for antigen binding and impart antigen binding specificity to the antibody. However, even one variable domain (or half of the Fv containing only three CDRs specific for the antigen), although less affinity than the entire binding site, has the ability to recognize and bind antigens.

"Single-chain Fv", also abbreviated as "sFv" or "scFv", is V _H linked by a single polypeptide chain. And an antibody fragment comprising a V _L antibody domain. The sFv polypeptide preferably further comprises a polypeptide linker between the V _H domain and the V _L domain, which allows the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994), Borrebaeck 1995, the following references.

The term "diabody" refers to the V _H domain and V _L SFv fragments (see paragraph above) with short linkers (about 5 to 10 residues) between domains were constructed to form interchain pairings rather than intrachain pairings of V domains, resulting in bivalent fragments, ie two antigen-binding sites Refers to a small antibody fragment prepared by generating a fragment. Bispecific diabodies include the V _H domain and V _L of two antibodies. Heterodimers whose domain consists of two “cross” sFv fragments present in different polypeptide chains. Diabodies are described in EP 404 097; WO 93/11161; And Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) and the like.

A “humanized” form of a non-human (eg rodent) antibody is a chimeric antibody that contains a minimal sequence derived from a non-human antibody. In most cases, a humanized antibody is a hypervariable region of a non-human species (donor antibody), such as a mouse, rat, rabbit or non-human primate, in which residues of the recipient's hypervariable region have the desired antibody specificity, affinity and ability. Human immunoglobulin (receptor antibody) replaced with a residue of. In some cases, the framework region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. In addition, the humanized antibody may comprise residues not found in the recipient antibody or the donor antibody. Such modifications further enhance antibody performance. Typically, a humanized antibody will comprise substantially all of one or more, typically two, variable domains, where all or substantially all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins and all or Virtually all FRs are FRs of human immunoglobulin sequences. In addition, the humanized antibody will optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For more details, see Jones et al., Nature 321: 522-525 (1986), Riechmann et al., Nature 332: 323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

A “species-dependent antibody”, eg, a mammalian anti-human IgE antibody, has a higher binding affinity for the antigen from the first mammalian species than the binding affinity for the homologue of the antigen from the second mammalian species. It is a strong antibody. Typically, a species-dependent antibody “specifically binds” to a human antigen (ie, its binding affinity (Kd) value is about 1 × 10 ⁻⁷ M or less, preferably about 1 × 10 ⁻⁸ M or less, most Preferably less than about 1 × 10 ⁻⁹ M), but the binding affinity for the homologue of the antigen from the second non-human mammal species is at least about 50 times or about 500 times the binding affinity for the human antigen. More than twice or about 1000 times weaker. The species-dependent antibody can be any of the various types of antibodies as defined above, but is preferably a humanized antibody or a human antibody.

A "TAT binding oligopeptide" is an oligopeptide that preferably binds specifically to a TAT polypeptide as described herein. TAT binding oligopeptides may be chemically synthesized using known oligopeptide synthesis or may be prepared and purified using recombinant techniques. Typically, the length of the TAT binding oligopeptide is about 5 or more amino acids, alternatively about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 Or 100 or more, such oligopeptides may preferably specifically bind to a TAT polypeptide as described herein. TAT binding oligopeptides can be identified without undue trial and error using known techniques. In this regard, it should be appreciated that screening techniques for oligopeptide libraries for oligopeptides that can specifically bind polypeptide targets are known in the art (see, eg, US Pat. Nos. 5,556,762, 5,750,373, 3D). 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143, PCT publications WO 84/03506 and W0 84/03564, Geysen et al., Proc. Natl Acad. Sci. USA, 81: 3998-4002 (1984), Geysen et al., Proc. Natl. Acad. Sci. USA, 82: 178-182 (1985), Geysen et al., In Synthetic Peptides as Antigens, 130-149 (1986), Geysen et al., J. Immunol.Meth., 102: 259-274 (1987), Schools et al., J. Immunol., 140: 611 -616 (1988), Cwirla, SE et al. (1990) Proc. Natl. Acad. Sci. USA, 87: 6378, Lowman, HB et al. (1991) Biochemistry, 30: 10832, Clackson, T. et al. (1991) Nature, 352: 624, Marks, JD et al. (1991), J. Mol. Biol., 222: 581, Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 8363 and Smith, G. P. (1991) Current Opin. Biotechnol., 2: 668).

A "TAT binding organic molecule" is an organic molecule that is not an oligopeptide or an antibody as defined herein that preferably binds specifically to a TAT polypeptide as described herein. TAT binding organic molecules can be identified and chemically synthesized using known methods (see, eg, PCT publications WO 00/00823 and WO 00/39585). Typically, the size of the TAT binding organic molecule is less than about 2000 Daltons, alternatively less than about 1500 Daltons, 750 Daltons, 500 Daltons, 250 Daltons or 200 Daltons, and is preferably specifically specific to the TAT polypeptide as described herein. Such organic molecules capable of binding can be identified without undue trial and error using known techniques. In this regard, it should be appreciated that techniques for screening organic molecular libraries for molecules capable of binding to polypeptide targets are known in the art (see, eg, PCT publications WO 00/00823 and WO 00/39585).

Antibodies, oligopeptides or other organic molecules that “bind” to an antigen of interest, eg, a tumor-associated polypeptide antigen target, bind with sufficient affinity to the antigen such that the antibody, oligopeptide or other organic molecule expresses the antigen. It is useful as a diagnostic and / or therapeutic agent in the targeting of cells and does not have significant cross-reaction with other proteins. In such embodiments, the degree of binding of the antibody, oligopeptide or other organic molecule with the "non-target" protein is determined by fluorescence activated cell sorter (FACS) analysis or radioimmunoprecipitation (RIA). Or less than about 10% of the binding of another organic molecule to its specific target protein. Antibodies, oligopeptides or other organic molecules that "specifically bind" or "specific" to a specific polypeptide or epitope on a particular polypeptide bind to a particular polypeptide or epitope on a particular polypeptide, but substantially to any other polypeptide or polypeptide epitope. Do not combine

Antibodies, oligopeptides or other organic molecules or “growth inhibiting” antibodies, oligopeptides or other organic molecules that “inhibit the growth of tumor cells expressing TAT polypeptide” can be measured by binding to cancer cells expressing or overexpressing the appropriate TAT polypeptide. It is to suppress growth. The TAT polypeptide may be a transmembrane polypeptide expressed on the surface of cancer cells or a polypeptide produced and secreted by cancer cells. Preferred growth inhibitory anti-TAT antibodies, oligopeptides or organic molecules have more than 20%, preferably about 20% to about 50%, even more preferably more than 50% growth of TAT-expressing tumor cells compared to appropriate controls ( For example from about 50% to about 100%), where the control group is typically a tumor cell that has not been treated with the antibody, oligopeptide or other organic molecule to be tested. In one embodiment, growth inhibition can be measured at an antibody concentration of about 0.1-30 μg / ml or about 0.5 nM to 200 nM in cell culture, wherein the growth inhibition is exposed to the antibody and 1 to 10 Measure after days. Inhibition of growth of tumor cells in vivo can be measured by various methods as described in the Examples section below. Proliferation of tumor size or tumor cells within about 5 days to 3 months, preferably about 5 to 30 days from the first administration of the antibody when administering from about 1 μg to about 100 mg of anti-TAT antibody per kg of body weight When this is reduced, the antibody is said to have a growth inhibitory effect in vivo.

Antibodies, oligopeptides, or other organic molecules that "induce apoptosis" may bind to annexin V, fragmentation of DNA, cell contraction, expansion of vesicles, cell fragmentation, and / or membrane vesicles (apoptosis). Induction of planned cell death, as measured by the formation of a sieve). Typically, such cells are cells that overexpress TAT. Preferably, the cells are tumor cells, for example prostate tumor cells, breast tumor cells, ovarian tumor cells, gastric tumor cells, endometrial tumor cells, lung tumor cells, kidney tumor cells, colon tumor cells, bladder tumor cells . Various methods can be used to investigate cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding, DNA fragmentation can be assessed through DNA laddering, and nucleation / chromosomal aggregation that occurs with DNA fragmentation is hypodyne. This can be confirmed by any increase in hypodiploid cells. Preferably, the antibody, oligopeptide or other organic molecule that induces apoptosis is about 2 to 50 times, preferably about 5 to 50 times, most preferably about 2 to 50 times anneal binding induction compared to untreated cells in annexin binding assays. Is about 10 to 50 times.

The "effector function" of an antibody refers to the biological activity attributable to the Fc region (natural sequence Fc region or amino acid sequence variant Fc region) of an antibody, and depends on the antibody isotype. Examples of antibody effector functions include C1q binding; Complement-dependent cytotoxicity; Fc receptor binding; Antibody-dependent cell mediated cytotoxicity (ADCC); Phagocytosis; Down regulation of cell surface receptors (eg B cell receptor) and B cell activation.

“Antibody-dependent cell mediated cytotoxicity” or “ADCC” refers to the secretion Ig bound to the Fc receptor (FcR) present on certain cytotoxic cells (eg, natural killer (NK) cells, neutrophils and macrophages). It refers to a cytotoxic form that allows cytotoxic effector cells to specifically bind to target cells bearing antigens and then kill the target cells with cytotoxins. Antibodies are "inorganic" of cytotoxic cells and are absolutely necessary for such cell death. NK cells, primary cells that mediate ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is described by Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991)], summarized in Table 3 on page 464. To assess ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed as described in US Pat. No. 5,500,362 or 5,821,337. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, such as in animal models as disclosed in Clynes et al., PNAS (USA) 95: 652-656 (1998) and the like.

"Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. Preferred FcRs are native sequence human FcRs. In addition, preferred FcRs are receptors (gamma receptors) that bind IgG antibodies, which include receptors of the FcγRI, FcγRII and FcγRIII subclasses, which include splice forms that differ from allelic variants of these receptors. do. FcγRII receptors mainly include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibiting receptor”), whose similar cytoplasmic domains have different amino acid sequences. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see M. in Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). ). For a review of FcRs, see Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991), Capel et al., Immunomethods 4: 25-34 (1994), and de Haas et al. , J. Lab. Clin. Med. 126: 330-41 (1995) Other FcRs, including those to be identified later, are included in the term “FcR” herein, which includes parental IgG to the fetus. Also delivered are neonatal receptor FcRn (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)).

"Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably such cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, but PBMCs and NK cells are preferred. Effector cells can be isolated from natural sources, such as blood.

"Complement-dependent cytotoxicity" or "CDC" refers to lysis of target cells in the presence of complement. The classical complement activation pathway is initiated by the binding of the first component (C1q) of the complement system to an antibody that binds the homologous antigen of the complement (an antibody of the appropriate subclass). To assess complement activation, see, eg, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) can be performed CDC analysis.

The terms "cancer" and "cancerous" refer to the physiological state of a mammal, which is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignant tumor. More specific examples of such cancers include lung cancer, peritoneal cancer, liver cancer, including squamous cell carcinoma (e.g. epithelial squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma of the lung, Stomach cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, Moon cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple melanoma and B-cell lymphoma, brain cancer as well as head and neck cancer and related metastases.

As used herein, "tumor" refers to all neoplastic cell growth and proliferation and to all pre-cancerous cells and cancerous cells and to cancerous and cancerous tissues, whether malignant or benign.

Antibodies, oligopeptides, or other organic molecules that "induce cell death" are those that die alive. The cell is a cell expressing a TAT polypeptide and is preferably a cell that overexpresses a TAT polypeptide as compared to normal cells of the same tissue type. The TAT polypeptide may be a transmembrane polypeptide expressed on the surface of cancer cells or a polypeptide produced and secreted by cancer cells. Preferably, the cells are cancer cells, for example breast cancer cells, ovarian cancer cells, gastric cancer cells, endometrial cancer cells, salivary gland cancer cells, lung cancer cells, kidney cancer cells, colon cancer cells, thyroid cancer cells, pancreatic cancer cells or bladder cancer cells. In vitro cell death is measured in the absence of complement and immune effector cells, and cell death by antibody-dependent cell mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) can be distinguished. Thus, cell death assays can be performed using heat-inactivated serum (ie, absence of complement) and in the absence of immune effector cells. To determine antibodies, oligopeptides or other organic molecules that can induce cell death, propidium iodide (PI), trypan blue (see Moore et al. Cytotechnology 17: 1-11 (1995)) or The extent of integrative damage of the membrane assessed by 7AAD uptake can be assessed in comparison to untreated cells. Preferred antibodies, oligopeptides or other organic molecules that induce cell death are those that induce PI uptake in PI uptake assays in BT474 cells.

A "TAT-expressing cell" is a cell expressing endogenous TAT or transfected TAT on the cell surface or in secreted form. A "TAT-expressing cancer" is a cancer comprising a cell having a TAT polypeptide on the cell surface or producing and secreting a TAT polypeptide. A "TAT-expressing cancer" may optionally express sufficient levels of TAT on its cell surface such that an anti-TAT antibody, oligopeptide or other organic molecule may bind to TAT on the surface of the cancer cell to produce a therapeutic effect against the cancer. . In other embodiments, a "TAT-expressing cancer" optionally produces and secretes a sufficient level of TAT polypeptide such that an anti-TAT antibody, oligopeptide or other organic molecular antagonist can bind to it to produce a therapeutic effect on the cancer. . For the latter, the antagonist may be an antisense oligonucleotide that reduces, inhibits or prevents the production and secretion of secreted TAT polypeptide by tumor cells. Cancers that "overexpress" a TAT polypeptide are cancers that express, produce and secrete TAT at significantly higher levels than non-cancerous cells of the same tissue type at their cell surface. Such overexpression may be caused by gene amplification or may be caused by increased transcription or translation. TAT polypeptide overexpression assesses an increase in the level of TAT protein present on or secreted by the cell surface (eg, isolated TAT polypeptide that can be prepared from isolated nucleic acids encoding TAT polypeptide using recombinant DNA techniques). Immunohistochemical analysis using an anti-TAT antibody against; evaluation through FACS analysis and the like). Alternatively or in addition, for example, fluorescent in situ hybridization using nucleic acid-based probes corresponding to TAT-encoding nucleic acids or their complements [FISH; WO 98/45479 (published Oct. 1998), TAT in cells via Southern blotting, Northern blotting or polymerase chain reaction (PCR) techniques such as real-time quantitative PCR (RT-PCR), etc. Levels of polypeptide-encoding nucleic acids or mRNA can be measured. TAT overexpression can also be studied, for example, by measuring free antigen in biological fluids such as serum using antibody-based assays (US Pat. No. 4,933,294, issued June 12, 1990). WO 91/05264 published April 18, 1991; US Pat. No. 5,401,638, issued March 28, 1995; and Sias et al., J. Immunol.Methods 132: 73- 80 (1990)]. Apart from the above assays, those skilled in the art can use various in vivo assays. For example, cells in a patient's body may optionally be exposed to an antibody labeled with a detectable label, such as a radioisotope, and whether such antibody binds to the patient's cells, for example, This can be confirmed by analyzing the biopsy taken from the patient prior to external scanning or exposure to the antibody.

As used herein, the term “immunoadhesin” refers to an antibody-like molecule that combines the effector function of an immunoglobulin constant domain and the binding specificity of a heterologous protein (“adhesin”). Structurally, immunoadhesin comprises a fusion of an immunoglobulin constant domain sequence with an amino acid sequence (ie, “heterologous”) having the desired binding specificity, but not the antigen recognition site and antigen binding site of the antibody. The adhesin portion of an immunoadhesin molecule is typically a contiguous amino acid sequence comprising at least the binding site of a receptor or ligand. Immunoglobulin constant domain sequences in immunoadhesin may be any such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM From immunoglobulins.

As used herein, the word “label” refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody, oligopeptide or other organic molecule to produce an “labeled” antibody, oligopeptide or other organic molecule. The label may itself be detectable (eg, a radioisotope label or fluorescent label) or, in the case of an enzyme label, may catalyze the chemical change of the detectable substrate compound or composition.

As used herein, the term “cytotoxic agent” refers to a substance that inhibits or interferes with the function of a cell and / or causes cell destruction. Such terms include radioisotopes (eg, radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and Lu); Chemotherapeutic agents such as methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents (intercalating) agent); Enzymes such as nucleolytic enzymes and fragments thereof; Antibiotic; And toxins such as small molecule toxins or enzymatically active toxins derived from bacteria, fungi, plants or animals (including fragments and / or variants thereof); And various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. Antitumor agents destroy tumor cells.

As used herein, "growth inhibitor" refers to a compound or composition that inhibits the growth of cells, particularly TAT-expressing cancer cells, in vitro or in vivo. Thus, the growth inhibitory agent may be one that significantly reduces the percentage of TAT-expressing cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at periods other than S phase), such as agents that induce G1 arrest and M arrest. Conventional M blockers include vincas (vincristine and vinblastine), taxanes and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. Examples of agents that cause S phase arrest in the after G1 arrest are DNA alkylating agents, for example tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and ara-C, and the like. have. For more information, see Murakami et al., The Molecular Basis of Cancer, Mendelsohn and Israel, eds., "Cell cycle regulation, oncogens, and antineoplastic drugs" (WB Saunders: Philadelphia, 1995), Chapter 1, especially 13 ]. Taxanes (paclitaxel and docetaxel) are both anticancer drugs derived from attention. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer) derived from European spotlight is packedclitaxel (TAXOL®, Bristol-Myers Squibb product) Is a semisynthetic analog of. Paclitaxel and docetaxel stabilize microtubules by promoting the assembly of tubulin dimers into microtubules and inhibiting depolymerization, thereby inhibiting mitosis of cells.

"Doxorubicin" is an anthracycline antibiotic. The full chemical name of doxorubicin is (8S-cis) -10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl) oxy] -7,8,9,10- Tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5,12-naphthasenedione.

The term “cytokine” is a generic name for proteins released by one cell population and acts as an intercellular mediator on another cell. Examples of such cytokines include lymphokines, monokines and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; Parathyroid hormone; Thyroxine; insulin; Proinsulin; Relaxine; Prorelaxin; Glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH); Liver growth factor; Fibroblast growth factor; Prolactin; Placental lactogen; Tumor necrosis factor-α and -β; Muller-inhibiting substance; Mouse gonadotropin-related peptide; Inhibin; Activin; Vascular endothelial growth factor; Integrin; Thrombopoietin (TPO); Nerve growth factors such as NGF-β; Platelet-growth factor; Transgenic growth factors (TGF) such as TGF-α and TGF-β; Insulin-like growth factor-I and -II; Erythropoietin (EPO); Osteoinductive factor; Interferons such as interferon-α, -β and -γ; Colony stimulating factor (CSF) such as macrophage-CSF (M-CSF); Granulocyte-macrophage-CSF (GM-CSF); And granulocyte-CSF (G-CSF); Interleukin (IL), for example IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 11, IL-12; Tumor necrosis factors such as TNF-α and TNF-β; And other polypeptide factors including LIF and kit ligands (KL). The term cytokine as used herein includes proteins and biologically active equivalents of native sequence cytokines from natural sources or from recombinant cell culture.

Typically, the term “packaging insert” is contained in a commercially available package of a therapeutic product and includes instructions containing information about the symptoms, usage, dosage, method of administration, contraindications, and / or warnings regarding the use of such therapeutic product. Used to speak.

II. Compositions and Methods of the Invention

A. Anti-TAT Antibodies

In one embodiment, the present invention provides anti-TAT antibodies that can be used herein as therapeutics and / or diagnostics. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific and heteroconjugate antibodies.

One. Polyclonal antibodies

Polyclonal antibodies are preferably produced in animals by multiple injections of the relevant antigen and adjuvant into the subcutaneous (sc) or intraperitoneal (ip). It may be useful to bind the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized. For example, difunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide esters (linking through cysteine residues), N-hydroxysuccinimides (through lysine residues), glutaraldehyde, succinic anhydride The antigen associated with a keyhole limpet hemocyanin (KLH), serum albumin, bovine tyroglobulin or soy trypsin inhibitor, using SOCl ₂ or R ¹ N = C = NR where R and R ¹ are different alkyl groups Can be combined.

Animals were treated with the antigen, immunogenicity by mixing 100 μg or 5 μg of the protein or conjugate (which is the dose for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and then injecting the solution intradermally into multiple sites. Immunize against conjugates or derivatives. One month later, the animal is boosted by subcutaneous injection of 1/5 to 1/10 of the initial amount of peptide or conjugate included in Freund's complete adjuvant into several sites. After 7-14 days, the animals are collected for analysis of antibody titers in serum. Boost the animal until the titer stabilizes. Conjugates can also be made as protein fusions in recombinant cell culture. In addition, coagulants such as alum are suitably used to enhance the immune response.

2. Monoclonal antibodies

Monoclonal antibodies can be prepared using the hybridoma method first described in Kohler et al., Nature, 256: 495 (1975) or by recombinant DNA methods (see US Pat. No. 4,816,567).

In the hybridoma method, mice or other suitable host animals (eg hamsters) are immunized as mentioned above to induce lymphocytes which can produce or produce antibodies that will specifically bind to the protein used for immunization. . Alternatively, lymphocytes can be immunized in vitro. After immunization, lymphocytes are isolated and lymphocytes are fused with myeloma cell lines using suitable fusing agents, such as polyethylene glycol, to form hybridoma cells. Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)].

The resulting hybridoma cells are grown by inoculation into a suitable culture medium which preferably contains one or more substances that inhibit the growth or survival of unfused parental myeloma cells (also called fusion partners). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for hybridomas is typically a substance that inhibits the growth of HGPRT-deficient cells. Hypoxanthine, aminopterin and thymidine (HAT medium).

Myeloma cells, which are preferred fusion partners, are cells that efficiently fuse and support stable high-level production of antibodies by selected antibody producing cells and are sensitive to the selection medium for selecting the myeloma cells from unfused parental cells. Preferred myeloma cell lines are derived from murine myeloma cell lines such as MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, San Diego, California USA), and SP-2 and derivatives, for example X63-Ag8-653 cells (available from American Type Culture Collection, Manassas, Virginia USA). Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies are also described in Kozbor, J. Immunol., 133: 3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987).

Culture medium in which hybridoma cells are grown is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is measured by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Measure with).

Binding affinity of monoclonal antibodies is described, for example, in Munson et al., Anal. Biochem., 107: 220 (1980), which can be determined by Scatchard analysis.

Once the hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned in a limited dilution method and grown by standard methods [Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)]. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, for example, hybridoma cells can be injected into mice to grow the hybridoma cells in vivo as ascites tumors in an animal.

Monoclonal antibodies secreted by the subclones are conventional antibody purification procedures, such as affinity chromatography (eg, using Protein A-Sepharose or Protein G-Sepharose), ion chromatography, hydroxylapatite It is suitable to separate from the culture medium, ascites fluid or serum by chromatography, gel electrophoresis or dialysis.

DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the murine antibody). . The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA is placed into an expression vector and then host cells, such as E. coli. Monoclonal antibodies can be synthesized in recombinant host cells by transfection into E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce antibody proteins. For recombinant expression in bacteria of DNA encoding the antibody, see Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-188 (1992).

In further embodiments, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) describes methods for separating murine and human antibodies, respectively, using phage libraries. The following publications describe the generation of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10: 779-783 (1992)) as well as strategies for constructing very large phage libraries. In vivo recombination and combinatorial infection methods are described by Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 (1993). Thus, these techniques are available alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

The DNA encoding the antibody can also be replaced with, for example, human heavy and light chain constant domain (C _H and C _L ) sequences instead of homologous murine sequences or described in US Pat. No. 4,816,567; And Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 (1984)], or all or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide) can be modified to produce a chimeric or fusion antibody polypeptide by linking it to an immunoglobulin coding sequence. . One antigen-binding site, which replaces the constant domain of the antibody with such a non-immunoglobulin polypeptide sequence, or the variable domain of one antigen-binding site of an antibody with these polypeptides, thereby exhibiting specificity for the antigen, and different Bivalent chimeric antibodies are generated that include another antigen-binding site that exhibits specificity for the antigen.

3. Human and Humanized Antibodies

In addition, the anti-TAT antibodies of the invention may comprise humanized antibodies or human antibodies. Humanized forms of non-human (eg, murine animal) antibodies may be immunoglobulin chains or fragments thereof (eg, Fv, Fab) comprising minimal sequences derived from chimeric immunoglobulins, non-human immunoglobulins , Fab ', F (ab') ₂ or other antigen binding subsequence of the antibody). Humanized antibodies are those in which the residues of the complementarity determining regions (CDRs) of the receptor are substituted with residues derived from CDRs of species other than the human (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and ability. Munoglobulins (receptive antibodies). In some cases, Fv framework residues of human immunoglobulins are substituted by corresponding non-human residues. Humanized antibodies may also comprise residues that are not found in the recipient antibody or in the CDR or framework sequences to be introduced. In general, humanized antibodies will comprise substantially all of one or more, generally two or more variable domains, where all or substantially all CDR regions correspond to regions of non-human immunoglobulins and all or substantially all FRs. The region corresponds to the region of the human immunoglobulin consensus sequence. In addition, humanized antibodies will comprise at least a portion of an immunoglobulin constant region (Fc), generally at least a portion of a human immunoglobulin region [Jones et al., Nature , 321 : 522-525 (1986); Riechmann et al., Nature , 332 : 323-329 (1988); And Presta, Curr . Op. Struct . Biol. 2 : 593-596 (1992).

Methods of humanizing nonhuman antibodies are well known in the art. In general, humanized antibodies incorporate one or more amino acid residues derived from non-human sources. These non-human amino acid residues are often referred to as "import" residues and are typically obtained from an "import" variable domain. Humanization consists essentially of methods such as Winter et al., Jones et al., Nature , 321 : 522-525 (1986), by substituting the corresponding sequences of human antibodies with rodent CDRs or CDR sequences. Riechmann et al., Nature , 332 : 323-327 (1988); Verhoeyen et al., Science , 239 : 1534-1536 (1988). Thus, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which substantially fewer sequences than one intact human variable domain are replaced by corresponding sequences from non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted with residues derived from similar sites of rodent antibodies.

The selection of human variable domains among the light and heavy chain variable domains is very important for reducing antigenic and HAMA responses (human anti-mouse antibodies) when the antibodies are used for human therapy. According to the so-called "best-fit" method, the entire library of known human variable domain sequences is screened for the sequences of the variable domains of rodent antibodies. Find human V domain sequences that are very similar to rodent V domain sequences and use the human framework region (FR) within these sequences to prepare humanized antibodies (Sims et al., J. Immunol. 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)). Another method uses a specific framework region derived from the consensus sequence of all human antibodies belonging to a particular subgroup of light or heavy chains. This same framework can be used to make several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol. 151: 2623 ( 1993).

It is also important to humanize the antibody to possess high binding affinity for the antigen and other beneficial biological properties. To achieve this object, according to a preferred method, humanized antibodies are prepared by methods of analysis of the sequences and various idealized humanized products using three-dimensional models of the sequences and humanized sequences. Three-dimensional immunoglobulin models are commonly used and are well known to those skilled in the art. Computer programs are available that illustrate and show possible three-dimensional conformations of selected candidate immunoglobulin sequences. Examination of this display allows for the analysis of possible roles of residues in the functionalization of candidate immunoglobulin sequences, ie the analysis of residues that affect the ability of candidate immunoglobulins to bind antigen. In this way, FR residues can be selected and combined from the accepting sequence and the import sequence to obtain the desired antibody properties, eg, increased affinity for the target antigen (s). In general, hypervariable region residues are directly and mostly substantially involved in influencing antigen binding.

Various forms of humanized anti-TAT antibodies are also contemplated. For example, the humanized antibody may be an antibody fragment, such as a Fab, optionally conjugated with one or more cytotoxic agent (s) to produce an immunoconjugate. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG ₁ antibody.

As an alternative to humanization, human antibodies can be generated. For example, a transgenic animal (eg, a mouse) can be produced that can produce the entire repertoire of human antibodies upon immunization in an environment free of exogenous immunoglobulin production. For example, deletion of the antibody heavy chain linkage region (J _H ) gene in chimeric and germline mutant mice completely inhibits endogenous antibody production. The introduction of human germline immunoglobulin gene sequences into such germline mutant mice will produce human antibodies upon antigen entry (Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993) Jakobovits et al., Nature, 362: 255-258 (1993); Bruggemann et al., Year in Immuno. 7:33 (1993); US Patent Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); 5,545,807; and WO 97/17852).

Alternatively, phage display technology (McCafferty et al., Nature 348: 552-553 [1990]) was used to extract human antibodies and antibody fragments from the immunoglobulin variable (V) domain gene repertoire of unimmunized donors in vitro. Can produce. According to this technique, antibody V domain genes are cloned in-frame into major or minor coat protein genes of fibrous bacteriophages such as M13 or fd and displayed as functional antibody fragments on the surface of phage particles. Since the fibrous particles contain a single stranded DNA copy of the phage genome, selection based on the functionality of the antibody enables selection of genes encoding antibodies that exhibit this functionality. Thus, phage show properties similar to some of the properties of B-cells. Phage display can be performed in a variety of formats, as described in Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3: 564-571 (1993). Several sources of V-gene fragments can be used for phage display. Clarkson et al. (Nature, 352: 624-628 (1991) isolated various types of anti-oxazolone antibodies from a small random combination library of V genes derived from the spleen of immunized mice. Repertoires of V genes derived from human donors can be constructed, and antibodies against various kinds of antigens (including autoantigens) can be prepared by Marks et al., J. Mol. Biol. 222: 581-597 (1991). ), Or Griffith et al., EMBO J. 12: 725-734 (1993); US Pat. Nos. 5,565,332 and 5,573,905).

As described above, human antibodies can also be produced by B cells activated in vitro (U.S. Patents 5,567,610 and 5,229,275).

4. Antibody Fragments

In certain circumstances, it is advantageous to use antibody fragments rather than whole antibodies. Smaller fragments can be removed quickly and have easier access to solid tumors.

Various techniques have been developed for generating antibody fragments. Traditionally, these fragments have been derived through proteolysis of intact antibodies [Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229: 81 (1985). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments are all E. coli. Expressed in E. coli. Since it can be secreted from E. coli, these antibodies can be easily produced in large quantities. Antibody fragments can be isolated from the antibody phage libraries described above. Or in the alternative, the Fab'-SH fragment is E. coli. It can be recovered directly from E. coli and chemically coupled to form F (ab ') ₂ fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another method, F (ab ') ₂ fragments can be isolated directly from recombinant host cell culture. Fab and F (ab ′ _{) 2} fragments that include salvage receptor binding epitope residues and have increased half-life in vivo are described in US Pat. No. 5,869,046. Other techniques for generating antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (svFv) [WO 93/16185; US Patent No. 5,571,894; And US Pat. No. 5,587,458. Since Fv and sFv are the only fragments with a binding site of the complete form without constant regions, they are suitable for reduced nonspecific binding during use in vivo. The sFv fusion protein can be constructed such that the effector protein is fused at the amino or carboxy terminus of sFv. For antibody manipulations, see Engineering, ed. Borrebaeck, supra. Such antibody fragments may also be "linear antibodies", eg, as described in US Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

5. Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. Exemplary bispecific antibodies may bind to two different epitopes of the TAT protein described herein. Others of these antibodies may include a TAT binding site along with a binding site for another protein. Alternatively, the anti-TAT arm may be a triggering molecule on white blood cells, such as a T-cell receptor molecule (eg, CD3), or an IgG (FcγRR) such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Can be combined with an arm that binds to the Fc receptor for) and can centralize and deploy cellular defense mechanisms against TAT-expressing cells. Bispecific antibodies can also be used to localize cytotoxic agents to cells that express TAT. These antibodies have TAT-binding arms and arms that bind to cytotoxic agents (eg, saporin, anti-interferon-α, vinca alkaloids, lysine A chain, methotrexate or radioisotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg, F (ab ') ₂ bispecific antibodies).

WO 96/16673 describes bispecific anti-ErbB2 / anti-FcγRIII antibodies and US Pat. No. 5,837,234 discloses bispecific anti-ErbB2 / anti-FcγRI antibodies. Bispecific anti-ErbB2 / Fcα antibodies are described in WO98 / 02463. US Pat. No. 5,821,337 describes bispecific anti-ErbB2 / anti-CD3 antibodies.

Methods of making bispecific antibodies are known in the art. Typically, the production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities [Millstein and Cuello, Nature , 305 : 537-539]. (1983). Due to the random classification of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, only one of which has an accurate bispecific structure. In general, the purification of the exact molecule carried out by an affinity chromatography step is rather cumbersome and the yield is low. Similar methods are described in WO 93/08829 (published May 13, 1993) and in Traunecker et al., EMBO J. , 10: 3655-3659 (1991).

According to another method, the variable domain (antibody-antigen binding site) of the antibody with the desired binding specificity can be linked to an immunoglobulin constant domain sequence. This linkage is preferably linked to an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, C _H 2 and C _H 3 regions. It is preferred that a first heavy chain constant region (C _H 1) comprising the site necessary for light chain binding is present in at least one of the fusions. DNA encoding an immunoglobulin heavy chain fusion, and optionally DNA encoding an immunoglobulin light chain, is inserted into a separate expression vector and co-transfected into a suitable host organism. This provides greater flexibility in controlling the mutual ratios of the three polypeptide fragments in embodiments where unequal proportions of the three polypeptide chains used in the production provide the optimal yield of the desired bispecific antibody. However, when two or more polypeptide chains having the same ratio are expressed in high yield or the ratio does not significantly affect the combined yield of the desired polypeptide chain, the coding sequences for the two or three polypeptide chains may be added to a single expression vector. Can be inserted.

In a preferred embodiment of this method, the bispecific antibody has a hybrid immunoglobulin heavy chain that exhibits a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain (which provides a second binding specificity) in the other arm. It consists of a pair of light chains. Since only half of the bispecific molecules have immunoglobulin light chains to facilitate separation, it has been found that this asymmetric structure facilitates the separation of desired bispecific compounds from unwanted immunoglobulin chain combinations. This method is described in (WO 94/04690). For more details on producing bispecific antibodies, see, eg, Suresh et al., Methods in Enzymmology , 121 : 210 (1986).

According to other methods disclosed in US Pat. No. 5,731,168, the interface between a pair of antibody molecules can be modified to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interface comprises at least a portion of the C _H 3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule were replaced with larger side chains (eg tyrosine or tryptophan). Replacement of large amino acid side chains with small amino acid side chains (eg, alanine or threonine) produced complementary "cavities" of the same or similar size for the large side chains at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers above the yield of other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked antibodies or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can couple to avidin and the other to biotin. Such antibodies have been proposed for use in targeting, for example, immune system cells to unwanted cells (US Pat. No. 4,676,980) and have also been proposed for the treatment of HIV infection (WO 91/00360, WO 92 /). 200373, and EP 03089). Heteroconjugate antibodies can be prepared using any convenient crosslinking method. Suitable crosslinkers are well known in the art and are described in US Pat. No. 4,676,980 with a number of crosslinking techniques.

Techniques for generating bispecific antibodies from antibody fragments are described in the literature. For example, bispecific antibodies can be prepared using chemical bonds. Brennan et al., Science 229: 81 (1985) describe a method of proteolytically intact antibodies to generate F (ab ') ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The Fab 'fragments generated were then converted to thionitrobenzoate (TNB) derivatives. Thereafter, one of the Fab'-TNB derivatives was reconverted to Fab'-thiol by reduction with mercaptoethylamine and mixed with equimolar amounts of other Fab'-TNB derivatives to form bispecific antibodies. The resulting bispecific antibodies can be used as substances for the selective immobilization of enzymes.

Due to the development of science and technology. The Fab'-SH fragments can be recovered directly from E. coli and chemically bound to form bispecific antibodies. Shalaby et al., J. Exp . Med . 175: 217-225 (1992) disclose the production of fully humanized bispecific antibody F (ab ') ₂ molecules. Each Fab 'fragment is E. coli. Bispecific antibodies were secreted from E. coli and linked via designated in vitro chemical coupling methods. The bispecific antibody thus formed was able to bind cells overexpressing the ErbB2 receptor and normal human T cells, as well as induce lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

In addition, several techniques are disclosed for making and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies were prepared using leucine zippers (Kostelny et al., J. Immunol . 148 (5): 1547-1553 (1992)). By gene fusion, leucine zipper peptides from Fos and Jun proteins were linked to the Fab 'portion of two different antibodies. The hinge region of the antibody homodimer was reduced to form a monomer and then reoxidized to form an antibody heterodimer. This method can also be used as a method for producing antibody homodimers. The "diabody" technique described by Hollinger et al., Proc . Natl . Acad . Sci . USA 90: 6444-6448 (1993) provides another mechanism for making bispecific antibody fragments. This fragment comprises a heavy chain variable domain (V _H ) linked by a linker to the light chain variable domain (V _L ), which is too short to pair the two domains on the same chain. Therefore, V _H and V _L domains of one fragment are made a complementary V _H and V _L domain pairs and of another fragment, thereby forming two antigen-binding sites. Another method of making bispecific antibody fragments using single chain Fv (sFv) dimers has been reported (see Gruber et al., J. Immunol . 152: 5368 (1994)).

Antibodies with antibody titers greater than two are also contemplated. For example, trispecific antibodies can also be prepared (Tutt et al., J. Immunol. 147: 60 (1991).

6. Heteroconjugate Antibodies

Heteroconjugate antibodies are also included within the scope of the present invention. Heteroconjugate antibodies consist of two covalently bound antibodies. Such antibodies can be used, for example, to target immune system cells to unwanted cells (US Pat. No. 4,676,980), and for the treatment of HIV infection (WO 91/00360, US 92/200373, and EP agents). 03089). It is contemplated that heteroconjugate antibodies can be prepared in vitro using methods known in synthetic protein chemistry, including methods using crosslinkers. For example, immunotoxins can be prepared by applying a disulfide exchange reaction or by forming a thioether bond. Suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutyrimidate, and reagents disclosed in US Pat. No. 4,676,980.

7. Multivalent Antibodies

Multivalent antibodies can internalize (and / or catalyze) more rapidly in cells that express the antigen to which the antibody binds than divalent antibodies. Antibodies of the invention may be multivalent antibodies having three or more antigen binding sites (classes other than the IgM class; e.g., tetravalent antibodies), which are produced by recombinant expression of a nucleic acid encoding a polypeptide chain of an antibody. It can be produced easily. Multivalent antibodies may comprise a dimerization domain and three or more antigen binding sites. Preferred dimerization domains comprise (or consist of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites at the amino-terminus of this region. Preferred multivalent antibodies herein comprise (from or consist of) three to about eight, preferably four antigen binding sites. The multivalent antibody comprises one or more polypeptide chains (preferably two polypeptide chains), wherein the polypeptide chain (s) comprises two or more variable domains. For example, the polypeptide chain (s) may comprise VD1- (Xl) _n -VD2- (X2) _n -Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain (s) can be a VH-CH 1-flexible linker-VH-CH 1 -Fc region chain; Or a VH-CH1-VH-CH1-Fc region chain. The multivalent antibodies herein preferably also comprise two or more (preferably four) light chain variable domain polypeptides. The multivalent antibody herein may comprise, for example, about 2 to about 8 light chain variable domain polypeptides. Light chain variable domain polypeptides contemplated herein include light chain variable domains and optionally also CL domains.

8. Effector function operation

It may be desirable to modify the antibodies of the invention for effector function to enhance, for example, the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC) of the antibody. This can be accomplished by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or additionally, cysteine residue (s) may be introduced into the Fc region to form interchain disulfide bonds in this region. Homodimeric antibodies thus produced may have improved internalization capacity and / or increased complement-mediated cell death and antibody-dependent intracellular cytotoxicity (ADCC) (Caron et al., J. Exp Med . , 176: 1191-1195 (1992) and Shopes, J. Immunol . , 148: 2918-2922 (1992)). Homodimeric antibodies with enhanced antitumor activity can also be prepared using the heterobifunctional crosslinks described in Wolfff et al. Cancer Research , 53 : 2560-2565 (1993). Alternatively, antibodies with double Fc regions can be engineered to enhance complement cytolysis and ADCC ability (see Stevenson et al., Anti-Cancer Drug Design , 3: 219-230 (1989)). ).

To increase the serum half-life of the antibody, salvage receptor binding epitopes can be introduced into, for example, antibodies (particularly antibody fragments) described in US Pat. No. 5,739,277. As used herein, “salvage receptor binding epitopes” refer to epitopes of the Fc region of IgG molecules (eg, IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ ) that serve to increase serum half-life in vivo of IgG molecules. Means.

9. Immunoconjugates

The invention also relates to cytotoxic substances, such as chemotherapeutic agents, growth inhibitors, toxins (eg, enzymatically active bacteria, fungi, plant or animal toxins, or fragments thereof) or radioactive isotopes (ie , An immunoconjugate comprising an antibody conjugated with a radioconjugate).

Chemotherapeutic agents useful for the production of such immunoconjugates are described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), lysine A chain, abrin A chain, modede Sin A chain, Alpha-sarsin, Alurites Fordi fordii ) protein, diantine protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, cursin, crotin, sapaonaria opininalis (sapaonaria officinalis) inhibitors, gelonin, mitogeline, restrictocin, phenomycin, enomycin, and trichothecene. Several radionuclides can be used for the preparation of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re.

Various difunctional protein-coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), difunctional derivatives of imidoesters (eg Dimethyl adipimidate HCL), active esters (e.g. disuccinimidyl suverate), aldehydes (e.g. glutaraldehyde), bis-azido compounds (e.g. bis (p- Azidobenzoyl) hexanediamine), bis-diazonium derivatives (eg bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (eg toluene 2,6-diisocyanate), and bis Active fluorine compounds (eg 1,5-difluoro-2,4-dinitrobenzene) are used to make conjugates of antibodies and cytotoxic agents. For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science , 238 : 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is a typical chelating agent for conjugating radionuclides and antibodies (see WO 94/11026). box).

Also contemplated herein are conjugates composed of antibodies and one or more small molecule toxins (eg, calicheamicin, maytansinoids, tricotene and CC1065, and derivatives of these toxins exhibiting toxicity).

Maytansine  And Maytansinoid

In one embodiment, an anti-TAT antibody (full length or fragment) of the invention is conjugated to one or more maytansinoid molecules.

Maytansinoids are mitotic inhibitors that act to inhibit tubulin polymerization. Maytansine is an East African shrub Maytenus serrata. serrata ) was first isolated from US Pat. No. 3,896,111. Thereafter, certain microorganisms have also been found to produce maytansinoids such as maytansinol and C-3 maytansinol esters (US Pat. No. 4,151,042). Synthetic maytansinol, derivatives and homologues thereof are described, for example, in US Pat. No. 4,137,230; 4,248,870; 4,248,870; 4,256,746; 4,256,746; 4,260,608; 4,260,608; No. 4,265,814; 4,294,757; 4,307,016; 4,307,016; No. 4,308,268; 4,308,269; No. 4,309,428; 4,313,946; 4,313,946; 4,315,929; 4,315,929; 4,317,821; 4,317,821; No. 4,322,348; No. 4,331,598; No. 4,361,650; No. 4,364,866; 4,424,219; 4,424,219; No. 4,450,254; 4,362,663; And 4,371,533, the contents of which are incorporated herein by reference.

Maytansinoid Antibodies Conjugate

In an attempt to improve the treatment rate of maytansine and maytansinoids, maytansine and maytansinoids were conjugated to antibodies that specifically bind tumor cell antigens. Immunoconjugates containing maytansinoids are described, for example, in US Pat. No. 5,208,020; 5,416,064; And European Patent EP 0 425 235 B1, the contents of which are incorporated herein by reference. Liu et al., Proc. Natl . Acad . Sci . USA 93 : 8618-8623 (1996) describes immunoconjugates comprising maytansinoids (named DM1) linked to monoclonal antibody C242 for human colorectal cancer. Such conjugates have been shown to exhibit potent cytotoxicity against cultured colon cancer cells and have shown antitumor activity in tumor growth assays in vivo. Chai et al., Cancer Research 52: 127-131 (1992) disclose that maytansinoids are conjugated with a murine antibody A7 that binds to an antigen on a human colon cancer cell line via a disulfide linker, or HER An immunoconjugate that is conjugated with another murine monoclonal antibody TA.1 that binds -2 / neu oncozin is described. The cytotoxicity of this TA.1-maytansinoid conjugate was tested in vitro on human breast cancer cell line SK-BR-3 expressing 3 × 10 ⁵ HER-2 surface antigens per cell. Such conjugate drugs showed a similar degree of cytotoxicity as free maytansinoid drugs, where cytotoxicity could be enhanced by increasing the number of maytansinoid molecules per antibody molecule. A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.

Anti-TAT Polypeptide Antibody- Maytansinoid Conjugate  ( Immunoconjugates )

Anti-TAT antibody-maytansinoid conjugates are prepared by chemically binding anti-TAT antibodies to maytansinoid molecules without significantly reducing the biological activity of the antibody or maytansinoid molecule. Although even one molecule of toxin / antibody is expected to increase cytotoxicity than when using an antibody that is not bound to maytansinoid, an average of three to four maytansinoid molecules bound per antibody will function as an antibody. Or to increase cytotoxicity of target cells without negatively affecting solubility. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are described, for example, in US Pat. No. 5,208,020, and in the other patents and nonpatent publications mentioned above. Preferred maytansinoids are maytansinol and maytansinol analogs, for example various maytansinol esters, in which the aromatic ring is modified or modified at other positions of the maytansinol molecule.

There are many linking groups known in the art to make antibody-maytansinoid conjugates, which are described, for example, in US Pat. No. 5,208,020 or EP Pat. No. 0 425 235 B1 and Chari et al. , Cancer Research 52: 127-131 (1992). Such linking groups include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups or esterase labile groups, as described in the aforementioned patents, with disulfide and thioether groups being preferred. Do.

Conjugates of the antibody and maytansinoid may be prepared by various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), difunctional derivatives of imidoesters (e.g. dimethyl adipimidate HCL), active esters (e.g. disuccinimidyl suverate ), Aldehydes (e.g. glutaraldehyde), bis-azido compounds (e.g. bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (e.g. bis- (p-diazoniumbenzoyl) -ethylene Diamine), diisocyanates (eg toluene 2,6-diisocyanate) and bis-active fluorine compounds (eg 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) which provides disulfide linkages [Carlsson et al., Biochem. J. 173: 723-737 (1978) and N-succinimidyl-4- (2-pyridylthio) pentanoate (SPP).

The linker may be attached to the maytansinoid molecule at various positions, depending on the type of link. For example, ester linkages can be formed by reaction with hydroxyl groups using conventional coupling techniques. This reaction can occur at the C-3 position with hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with hydroxyl group, and the C-20 position with hydroxyl group. In a preferred embodiment, this linkage is formed at the C-3 position of maytansinol or a maytansinol homologue.

Calicheamicin

Still other immunoconjugates of interest include anti-TAT antibodies that are bound to one or more calicheamicin molecules. The calicheamicin family of antibiotics can cleave double stranded DNA at sub-picomole concentrations. For the preparation of conjugates of the calicheamicin family, U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all American Ciana) Meade's patent). Structural homologues of calicheamicin that may be used include, but are not limited to, γ ₁ ^I , α ₂ ^I , α ₃ ^I , N-acetyl-γ ₁ ^I , PSAG and θ ₁ ^I (Hinman et al. Cancer Research 53: 3336-3342 (1993), Lode et al. Cancer Research 58: 2925-2928 (1998); all of these US patents are patents of American Cyanamide. Another anti-tumor drug that can be conjugated with the antibody is QFA, which is an antifolate. There are sites of intracellular action on both calicheamicin and QFA, and they do not readily cross the plasma membrane. Therefore, uptake of these drugs intracellularly through antibody mediated internalization greatly enhances their cytotoxic effects.

Etc Cytotoxic agents

Other antitumor agents that can be conjugated to the anti-TAT antibodies of the invention include, in general, LL-E33288, described in BCNU, streptazocin, vincristine and 5-fluorouracil, US Pat. Nos. 5,053,394, 5,770,710. Group of agents known as binders, as well as esperamicin (US Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa ), lysine A chain, abrin A chain, modeine A chain, alpha-sarsin, alluret Fordy fordii ) protein, diantine protein, phytolacca americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, cursin, crotin, sapaonaria officinalis Inhibitors, gelonin, mitogeline, restrictocin, phenomycin, enomycin and tricortesene (see WO 93/21232 published October 28, 1993).

The present invention also contemplates immunoconjugates formed between an antibody and a compound that exhibits nucleolytic activity (eg, DNA endonucleases such as ribonucleases or deoxyribonucleases; DNases).

For selective destruction of the tumor, the antibody may contain highly radioactive atoms. Various radioisotopes can be used to generate anti-TAT antibodies bound to radioisotopes. Examples of radioisotopes include radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu. When the conjugate is used for diagnosis, the conjugate is a spin label for radioactive atoms for scintogram imaging studies, for example tc ^99m or I ¹²³ , or nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging (MRI)). For example, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Radiolabels or other labels can be introduced to the conjugates by known methods. For example, peptides can be biosynthesized or synthesized, for example, by chemical amino acid synthesis using suitable amino acid precursors with fluorine-19 instead of hydrogen. Labels such as tc ^99m or I ¹²³ , Re ¹⁸⁶ , Re ¹⁸⁸ and In ¹¹¹ can be attached to the peptide via cysteine residues. Yttrium-90 may be attached via lysine residues. Iodine-123 can be introduced using the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57). Another method is described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

Immunoconjugates composed of antibodies and cytotoxic agents include various bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), difunctional derivatives of imidoesters (e.g. dimethyl adipimidate HCL), active esters (e.g. disuccinimidyl suverate ), Aldehydes (eg glutaraldehyde), bis-azido compounds (eg bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (eg bis- (p-diazoniumbenzoyl) -ethylene Diamine), diisocyanates (eg toluene 2,6-diisocyanate) and bis-active fluorine compounds (eg 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins are described in Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for binding antibodies and radionucleotides (see WO 94/11026). . The linker may be a “cleavable linker” that facilitates the release of cytotoxic drugs in the cell. For example, acid labile linkers, peptidase-sensitive linkers, photo-labile dimethyl linkers or disulfide containing linkers can be used (Chari et al. Cancer Research 52: 127-131 (1992); US Pat. No. 5,208,020). Reference).

Alternatively, fusion proteins comprising anti-TAT antibodies and cytotoxic agents can be prepared, for example, by recombinant techniques or peptide synthesis. The length of the DNA may comprise two regions of the conjugate adjacent to each other, or each region encoding two regions of the conjugate separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate. .

In another embodiment, the antibody can be bound to a "receptor" (eg, streptavidin) for use in tumor pretargeting, wherein such antibody-receptor conjugate is administered to the patient and then removed from the circulatory system using a scavenger. After removal of the unbound conjugate, an "ligand" (eg avidin) that is bound to a cytotoxic agent (eg radionucleotide) is administered.

10. Immunoliposomes

The anti-TAT antibodies disclosed herein may also be formulated with immunoliposomes. “Liposomes” are small vesicles composed of various types of lipids, phospholipids, and / or surfactants useful for delivering drugs to mammals. The components of the liposomes are typically arranged in a bilayer formation arrangement similar to the lipid arrangement of the biofilm. Liposomes comprising this antibody can be prepared using methods known in the art, for example, Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); US Pat. Nos. 4,485,045 and 4,544,545 and WO 97/38731 (published October 23, 1997). Liposomes with increased circulation time are disclosed in US Pat. No. 5,013,556.

Particularly useful liposomes can be produced by reverse phase evaporation using lipid compositions containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are pushed through filters of defined pore size to yield liposomes with the desired diameter. Fab 'fragments of the antibodies of the invention can be conjugated to liposomes via disulfide-interchange reactions as described in Martin et al., J. Biol . Chem . , 257 : 286-288 (1982). have. Optionally, chemotherapeutic agents are included in the liposomes (see Gabriel et al., J. National Cancer Inst . , 81 (19): 1484 (1989)).

B. TAT Binding Oligopeptides

The TAT binding oligopeptides of the present invention are preferably oligopeptides which specifically bind to the TAT polypeptides described herein. TAT binding oligopeptides can be chemically synthesized using known oligopeptide synthesis methods or prepared and purified using recombinant techniques. TAT binding oligopeptides are generally at least about 5 amino acids in length, or about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 , 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 It is at least two in length and this oligopeptide is preferably capable of binding, in particular, to a TAT polypeptide as described herein. TAT binding oligopeptides can be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides that can specifically bind polypeptide targets are well known in the art. See, for example, US Pat. Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; And PCT Publication Nos. WO 84/03506 and WO84 / 03564; Geysen et al., Proc. Natl. Acad. Sci. USA, 81: 3998-4002 (1984); Geysen et al., Proc. Natl. Acad Sci. USA, 82: 178-182 (1985); Geysen et al., In Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol.Meth., 102: 259-274 ( 1987); Schoofs et al., J. Immunol., 140: 611-616 (1988), Cwirla, SE et al. (1990) Proc. Natl. Acad. Sci. USA, 87: 6378; Lowman, HB et al (1991) Biochemistry, 30: 10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, JD et al. (1991), J. Mol. Biol., 222: 581; Kang, AS et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 8363, and Smith, GP (1991) Current Opin. Biotechnol., 2: 668).

In this regard, bacteriophage (phage) display is a well known technique that allows screening large oligopeptide libraries to identify member (s) of the library that can specifically bind to polypeptide targets. Phage display is a technique in which variant polypeptides appear as fusion proteins for coat proteins on the surface of bacteriophage particles (Scott, J.K. and Smith, G. P. (1990) Science 249: 386). The utility of phage display lies in the fact that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be quickly and effectively aligned with respect to sequences that bind to target molecules with high affinity. Phage-like peptide (Cwirla, SE et al. (1990) Proc. Natl. Acad. Sci. USA, 87: 6378) or protein (Lowman, HB et al. (1991) Biochemistry, 30: 10832; Clackson, T. et. (1991) Nature, 352: 624; Marks, JD et al. (1991), J. Mol. Biol., 222: 581; Kang, AS et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8363) Displays of libraries have been used to screen millions of polypeptides or oligopeptides for those with specific binding properties (Smith, GP (1991) Current Opin. Biotechnol., 2: 668). Aligning phage libraries of random mutants requires strategies for preparing and propagating multiple variants, methods for affinity purification using target receptors, and means for assessing the consequences of increased binding [US Pat. No. 5,223,409, 5,403,484, 5,571,689 and 5,663,143.

Although most phage display methods utilize fibrous phage, lambda phage display systems (WO 95/34683; US 5,627,024), T4 phage display systems (Ren, ZJ. Et al. (1998) Gene 215: 439; Zhu, Z (1997) CAN 33: 534; Jiang, J. et al. (1997) can 128: 44380; Ren, ZJ. Et al. (1997) CAN 127: 215644; Ren, ZJ. (1996) Protein Sci. 5 Efimov, VP et al. (1995) Virus Genes 10: 173) and T7 phage display systems (Smith, GP and Scott, JK (1993) Methods in Enzymology, 217, 228-257; US 5,766,905) are also known. have.

Many other improvements and modifications to the basic phage display concept have been developed to date. These improvements have increased the ability of the display system to screen functional proteins for the desired properties and the ability of the display system to screen peptide libraries that bind selected target molecules. Combination reaction devices for phage display reactions have been developed (WO 98/14277), biphasic interactions using phage display libraries (WO 98/20169; WO 98/20159) and properties of inhibited helix peptides (WO 98/20036) ) Were analyzed and adjusted. WO 97/35196 provides a method for selectively isolating binding ligands by contacting a phage display library with a first solution in which the ligand binds to the target molecule and a second solution in which the affinity ligand does not bind to the target molecule to isolate the affinity ligand. The method is described. WO 97/46251 discloses the use of affinity purified antibodies for biopanning a random phage display library, followed by isolation of binding phage, followed by high affinity binding phage by a micropanning method using microplate wells. Describe how to isolate. Reported use of Staphylococcus aureus protein A as an affinity tag (Li et al. (1998) Mol Biotech., 9: 187). WO 97/47314 describes the use of substrate exclusion libraries to distinguish enzyme specificities using combinatorial libraries, which may be phage display libraries. Methods for selecting suitable enzymes for use as detergents using phage display are described in WO 97/09446. Other methods of selecting specific binding proteins are described in US Pat. No. 5,498,538, US Pat. No. 5,432,018, and WO 98/15833.

Methods for generating peptide libraries and screening such libraries are also described in US Pat. Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018, 5 5,698,426, 5,763,192 and 5,723,323.

C. TAT binding organic molecules

TAT binding organic molecules are preferably organic molecules different from oligopeptides or antibodies defined herein that specifically bind to the TAT polypeptides described herein. TAT binding organic molecules can be identified and chemically synthesized using known methods (see, eg, PCT Publications WO 00/00823 and WO 00/39585). TAT binding organic molecules are generally less than about 2000 Daltons in size, or less than about 1500, 750, 500, 250, or 200 Daltons, and are preferably specifically described herein, without unnecessary experimentation, using well known techniques. Identify organic molecules that can bind to In this regard, it is noted that techniques for screening organic molecular libraries for molecules capable of binding polypeptide targets are well known in the art (eg, PCT Publication WO 00/00823 and WO 00/39585). Reference). TAT binding organic molecules are for example aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, Thiols, thioethers, disulfides, carboxylic acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates , Aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolins, enamines, sulfonamides, epoxides, aziridine, isocyanates, sulfonyls Chloride, diazo compound or acid chloride and the like.

D. Screening of Anti-TAT Antibodies, TAT Binding Oligopeptides and TAT Binding Organic Molecules with Desired Properties

Techniques for generating antibodies, oligopeptides and organic molecules that bind to TAT polypeptides are described above. If desired, antibodies, oligopeptides or other organic molecules exhibiting specific biological characteristics can be further selected.

Growth inhibitory effects of the anti-TAT antibodies, oligopeptides or other organic molecules of the invention can be achieved by methods known to those skilled in the art, for example, using cells expressing TAT either implicitly or after transfection with the TAT gene. It can be measured. For example, suitable tumor cell lines and TAT-transfected cells are treated with various concentrations of the anti-TAT monoclonal antibodies, oligopeptides or other organic molecules of the invention for several days (eg 2-7 days) and crystal violet. Or stained with MTT or analyzed by several other colorimetric assays. Another method of measuring proliferation is to compare ³ H-thymidine uptake by cells treated under the presence or absence of an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule of the invention. After treatment, the cells are collected and the amount of radioactivity incorporated into the DNA is quantified with a scintillation counter. Suitable positive controls include cell lines treated with growth inhibitory antibodies known to inhibit the growth of selected cell lines. Growth inhibition of tumor cells in vivo can be measured in various ways known in the art. Preferably, the tumor cell is a cell that overexpresses a TAT polypeptide. Preferably, the anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule is about 25-100%, more preferably about 30 as compared to untreated tumor cells at an antibody concentration of about 0.5 to 30 μg / ml -100%, even more preferably about 50-100% or 70-100% will inhibit cell proliferation of TAT-expressing tumor cells in vitro or in vivo. Growth inhibition can be measured at an antibody concentration of about 0.5 to 30 μg / ml or about 0.5 nM to 200 nM in cell culture, wherein the growth inhibition is measured 1-10 days after exposure of the tumor cells to the antibody. Growth of tumor size or tumor cells within about 5 to 3 months, preferably about 5 to 30 days, from the first administration of the antibody when about 1 μg to about 100 mg of anti-TAT antibody per kg body weight is administered When this is reduced, the antibody is said to have a growth inhibitory effect in vivo.

In order to screen for anti-TAT antibodies, TAT binding oligopeptides or TAT binding organic molecules that induce cell death, the integrity of the membrane, as indicated by, for example, propidium iodide (PI), trypan blue or 7AAD uptake, The extent of damage can be assessed by comparison with the control. PI uptake assays can be performed in the absence of complement and immune effector cells. TAT polypeptide-expressing tumor cells are incubated with the medium alone or with a medium containing, for example, about 10 μg / ml of a suitable anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule. The cells are incubated for 3 days. After each treatment, the cells are washed and divided into 35 mm strainer-capped 12 x 75 tubes (1 ml per tube, 3 tubes per treatment group) to remove cell mass. Subsequently, PI (10 μg / ml) is added to the tube. Samples can be analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (Becton Dickinson). Anti-TAT antibodies, TAT binding oligopeptides or TAT binding organic molecules that induce statistically significant levels of cell death, as measured by PI uptake, may be used to induce cell death induction anti-TAT antibodies, TAT binding oligopeptides or TAT binding. It can be selected as an organic molecule.

For screening antibodies, oligopeptides or other organic molecules that bind to epitopes on TAT polypeptide bound by the desired antibody, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) Conventional cross-blocking analysis as can be performed. This assay can be used to determine whether the test antibody, oligopeptide or other organic molecule binds to the same site or epitope as known anti-TAT antibodies. Alternatively, or in addition, epitope mapping can be performed by methods known in the art. For example, antibody sequences can be mutated in a manner such as alanine scanning to identify contact residues. Mutant antibodies are first tested for binding with polyclonal antibodies to ensure that they are properly folded. Alternatively, peptides corresponding to various regions of the TAT polypeptide can be used in competition assays with the test antibody, or with characterized epitopes or antibodies with known epitopes and test antibodies.

Mediated by antibody-dependent enzyme E. Prodrug Therapy (ADEPT)

Antibodies of the invention can also be used in ADEPT by binding the antibody to a prodrug activating enzyme that converts a prodrug (eg peptidyl chemotherapeutic agent; see WO 81/01145) into an active anticancer agent [eg, WO 88/07378 and US Pat. No. 4,975,278.

Enzyme components of immunoconjugates useful for ADEPT include enzymes that can act on prodrugs in a way that converts them into a higher active cytotoxic form than the prodrug.

Enzymes useful in the methods of the invention include alkaline phosphatase useful for converting phosphate containing prodrugs into free drugs; Arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; Cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anticancer agent, 5-fluorouracil; Proteases useful for converting peptide-containing prodrugs into free drugs, such as seratia proteases, thermolysine, subtilisin, carboxypeptidase and cathepsin (eg cathepsin B and L); D-alanylcarboxypeptidase useful for converting prodrugs containing D-amino acid substituents; Carbohydrate cleavage enzymes such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; β-lactamase useful for converting drugs derivatized with β-lactams into free drugs; And penicillin amidase, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized with phenoxyacetyl or phenylacetyl groups in amine nitrogen to free drugs, respectively. . On the other hand, antibodies exhibiting enzymatic activity, also known in the art as "abzyme", can be used to convert prodrugs of the present invention to free active drugs [Massey, Nature 328: 457- 458 (1987). Antibody-abzyme conjugates can be prepared as described herein and delivered to a tumor cell population.

The enzymes of the present invention can be covalently linked to anti-TAT antibodies by techniques well known in the art, such as using the hetero-bifunctional crosslinkers discussed above. Alternatively, a fusion protein comprising at least the antigen binding region of an antibody of the invention linked to at least the functionally active site of the enzyme of the invention can be constructed using recombinant DNA techniques well known in the art [Neuberger et. al., Nature 312: 604-608 (1984).

F. Full Length TAT Polypeptides

The present invention also provides newly identified and isolated nucleotide sequences encoding polypeptides referred to herein as TAT polypeptides. Specifically, cDNAs (partial and full length cDNAs) encoding various TAT polypeptides have been identified and isolated as described in more detail in the Examples below.

Various cDNA clones were deposited in the ATCC as described in the Examples below. The actual nucleotide sequence of these clones can be readily determined by one skilled in the art by sequencing the clones deposited using routine methods in the art. Prospective amino acid sequences can be determined from nucleotide sequences using routine techniques in the art. For TAT polypeptides and coding nucleic acids described herein, in some cases we have found what is the best reading frame that can be identified using the sequence information available at the time.

G. Anti-TAT Antibody and TAT Polypeptide Variants

 In addition to the anti-TAT antibodies and full-length native sequence TAT polypeptides described herein, it is contemplated that anti-TAT antibodies and TAT polypeptide variants can be prepared. Anti-TAT antibodies and TAT polypeptide variants can be prepared by introducing appropriate nucleotide changes into the coding DNA and / or synthesizing the desired antibody or polypeptide. Those skilled in the art will appreciate that amino acid changes, such as a change in the number or position of glycosylation sites or a change in membrane anchoring properties, can alter the post-translational process of an anti-TAT antibody or TAT polypeptide.

Variants of the anti-TAT antibodies or TAT polypeptides described herein can be prepared using, for example, conservative and nonconservative mutation techniques and instructions disclosed in US Pat. No. 5,364,934. The variation may be a substitution, deletion or insertion of one or more codons encoding an anti-TAT antibody or TAT polypeptide in which the amino acid sequence of the anti-TAT antibody or TAT polypeptide is changed as compared to the native sequence anti-TAT antibody or TAT polypeptide. Optionally, mutations are made by replacing one or more amino acids with any other amino acid in one or more domains of one or more anti-TAT antibodies or TAT polypeptides. Whether an amino acid residue can be inserted, substituted or deleted without adversely affecting the desired activity is determined by comparing the sequence of the anti-TAT antibody or TAT polypeptide with that of a known homologous protein molecule and the amino acid produced in the region of high homology. This can be determined by minimizing the number of sequence changes. Amino acid substitutions may be the result of substitution of one amino acid with another amino acid having similar structural and / or chemical properties, eg, replacement of leucine with serine, ie, conservative substitution of amino acids. Insertion or deletion may optionally occur at about 1-5 amino acids. Acceptable variations can be determined by systematically inserting, deleting, or replacing amino acids in the sequence and testing the activity of the resulting variants represented by the full length or mature native sequence.

The application provides anti-TAT antibody or TAT polypeptide fragments. For example, when compared to full-length natural antibodies or proteins, these fragments may be truncated at the N- or C-terminus or deleted internal residues. Some fragments do not have amino acid residues that are not essential for the desired biological activity of the anti-TAT antibody or TAT polypeptide of the invention.

Anti-TAT antibodies and TAT polypeptide fragments can be prepared by any of a number of conventional techniques. Desired peptide fragments can be synthesized chemically. Other methods include enzymatic digestion methods, for example anti-TAT, by treating the protein with an enzyme known to cut the protein at sites defined by specific amino acid residues, or by cutting this DNA with a suitable restriction enzyme and isolating the desired fragment. Generating an antibody or TAT polypeptide fragment. However, another suitable technique includes isolating DNA fragments encoding the desired polypeptide fragments and amplifying by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are used as 5 'and 3' primers in PCR. Preferably, the anti-TAT antibody or TAT polypeptide fragment shares one or more biological and / or immunological activities with the native anti-TAT antibody or TAT polypeptide disclosed herein.

In specific embodiments, conservative substitutions of the target are shown in Table 6 under the heading of preferred substitutions. If the biological activity is changed by this substitution, the product was screened with more substantial changes, designated as substitutions in Table 6 below or described in more detail below with respect to amino acid species.

TABLE 6

Substantial modifications of the function or immunological identity of the anti-TAT antibody or TAT polypeptide may be achieved by (a) maintaining the structure of the polypeptide backbone at the substitution region, eg, in the form of a sheet or helix, or (b) a molecule at the target site. Or by selecting a substitution that significantly alters its effect of maintaining the volume of the side chain (c). Naturally occurring residues are divided into the following groups according to common side chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues affecting chain orientation: gly, pro; And

(6) aromatic; trp, tyr, phe.

Non-conservative substitutions will replace said one type of component with another type. In addition, the residues so substituted may be introduced at conservative substitution sites or more preferably at the remaining (non-conserved) sites.

Mutations can be made using methods known in the art such as oligonucleotide mediated (positioning) mutagenesis, alanine scanning and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl . Acids Res. , 13 : 4331 (1986); Zoller et al., Nucl . Acids Res. , 10 : 6487 (1987)], cassette mutagenesis [Wells et al., Gene , 34 : 315 (1985)], restriction selection mutagenesis [Wells et al., Philos . Trans. R. Soc . London SerA , 317 : 415 (1986)] or other known techniques can be performed on cloned DNA to produce an anti-TAT antibody or TAT polypeptide variant DNA of the invention.

Scanning amino acid assays can also be used to identify one or more amino acids along adjacent sequences. Preferred scanning amino acids are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Typically, alanine is a preferred scanning amino acid because it is less likely to remove side chains outside the beta-carbon and alter the backbone arrangement of the variants (Cunningham and Wells, Science , 244 : 1081-1085 (1989)). Alanine is also preferred because it is usually the most common amino acid. In addition, alanine is frequently found both in buried and exposed locations [Creighton, The Proteins , (WH Freeman & Co., NY); Chothia, J. Mol . Biol . , 150 : 1 (1976). If alanine substitutions do not produce adequate amounts of variants, isotopic amino acids can be used.

Any cysteine residue that is not involved in maintaining the proper conformation of the anti-TAT antibody or TAT polypeptide can generally be substituted with serine to improve the oxidative stability of the molecule and prevent false crosslinking. Conversely, cysteine binding (s) can be added to the anti-TAT antibody or TAT polypeptide to improve its stability (especially when the antibody is an antibody fragment such as an Fv fragment).

Particularly preferred types of substitutional variants include substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). In general, the product variant (s) chosen for further development will exhibit improved biological properties compared to the parent antibody that produced them. A convenient way to generate such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (eg 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The resulting antibody variants are displayed in a monovalent manner from filamentous phage particles as fusions with the gene III product of M13 packaged within each particle. Phage-display variants are then screened that exhibit the biological activity (eg binding affinity) of the phage-display variants as described herein. To identify candidate hypervariable region sites suitable for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be advantageous to analyze the crystal structure of the antigen-antibody conjugate to identify the point of contact between the antibody and human TAT polypeptide. Such contact residues and neighboring residues are candidates for substitution according to the techniques reviewed herein. Once such variants are generated, a panel of variants can be screened as described herein and further developed by selecting antibodies that exhibit good properties in one or more related assays.

Nucleic acid molecules encoding amino acid sequence variants of anti-TAT antibodies are prepared by a variety of methods known in the art. These methods include isolation methods from natural sources (for naturally occurring amino acid sequence variants) or oligonucleotide-mediated (or positional) mutagenesis, PCR mutagenesis, and previously prepared variants or non-mutants of anti-TAT antibodies. Methods of making by cassette mutagenesis include, but are not limited to.

H. Modifications of Anti-TAT Antibodies and TAT Polypeptides

Covalent modifications of anti-TAT antibodies and TAT polypeptides are within the scope of this invention. One form of covalent modification involves reacting a target amino acid residue of an anti-TAT antibody or TAT polypeptide with an organic derivatizing agent capable of reacting with a selected side chain or N- or C-terminal residue of the anti-TAT antibody or TAT polypeptide. . Derivatization with a bifunctional agent is useful for crosslinking or crosslinking an anti-TAT antibody or TAT polypeptide to a water-insoluble support matrix or surface, for example for use in anti-TAT antibody purification methods. Commonly used crosslinking agents are for example 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example esters with 4-azidosalicylic acid, Homo-functional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis (succinimidylpropionate), difunctional maleimides such as bis-N-maleimido-1,8-octane and Materials such as methyl-3-[(p-azidophenyl) dithio] propioimidate.

Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, lysine, Methylation of alpha-amino groups of arginine and histidine side chains [TE Creighton, Proteins: Structure and Molecular Properties , WH Freeman & Co., San Francisco, pp. 79-86 (1983), acetylation of N-terminal amines and amidation of C-terminal carboxyl groups.

Other types of covalent modifications of anti-TAT antibodies or TAT polypeptides within the scope of the present invention include changes in the natural glycosylation pattern of the antibody or polypeptide. For the purposes herein, a "change in natural glycosylation pattern" refers to the deletion of one or more carbohydrate moieties found in a native sequence anti-TAT antibody or TAT polypeptide (removal of potential glycosylation sites or by chemical and / or enzymatic methods). By means of deletion of glycosylation) and / or the addition of one or more glycosylation sites that are not present in the native sequence anti-TAT antibody or TAT polypeptide. The term also encompasses qualitative changes in glycosylation of natural proteins, including changes in the properties and proportions of the various carbohydrate residues present.

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to a carbohydrate moiety attached to the side chain of an asparagine moiety. Tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for attaching carbohydrate moieties to asparagine side chains using enzymes. Thus, the presence of one of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation means attaching one of sugar N-acetylgalactosamine, galactose or xylose to hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine You can also use

The addition of glycosylation sites to the anti-TAT antibody or TAT polypeptide is conveniently accomplished by changing the amino acid sequence to include one or more of the tripeptide sequences described above (for N-linked glycosylation sites). The change can be made by addition or substitution of one or more serine or threonine residues in the sequence of the original anti-TAT antibody or TAT polypeptide (for O-linked glycosylation sites). The amino acid sequence of the anti-TAT antibody or TAT polypeptide may be optionally altered through changes in the DNA level by mutating DNA encoding the anti-TAT antibody or TAT polypeptide at a preselected base to produce a codon that is translated into the desired amino acid. Can be.

Another means of increasing the number of carbohydrate residues on an anti-TAT antibody or TAT polypeptide is to couple the glycoside chemically or enzymatically to the polypeptide. Such methods are described, for example, in WO 87/05330 published September 11, 1987 and in Aplin and Wriston, CRC Crit . Rev. Biochem . , pp. 259-306 (1981).

Removal of carbohydrate residues present in an anti-TAT antibody or TAT polypeptide can be accomplished chemically or by substitution by mutation of a codon encoding an amino acid residue that functions by an enzyme or as a glycosylation target. Chemical deglycosylation techniques are known in the art and are described, for example, in Hakimuddin, et al., Arch. Biochem. Biophys . , 259 : 52 (1987) and Edge et al., Anal. Biochem . , 118 : 131 (1981). Enzymatic cleavage of carbohydrate residues on polypeptides can be accomplished using a variety of endoglycosidases and exoglycosidases [Thotakura et al., Meth . Enzymol . , 138 : 350 (1987).

Other types of covalent modifications of anti-TAT antibodies or TAT polypeptides are described in various ways in the manner described in US Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337. Linking the antibody or polypeptide to one of a nonproteinaceous polymer such as polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene. Antibodies or polypeptides are also prepared by, for example, coacervation techniques or interfacial polymerization reactions (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively). In the form of a colloidal drug delivery system (eg, liposomes, albumin microspheres, microemulsions, nano-particles and nano-capsules) or macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

In addition, the anti-TAT antibodies or TAT polypeptides of the invention can be modified in such a way as to form chimeric molecules comprising anti-TAT antibodies or TAT polypeptides fused to other heterologous polypeptides or amino acid sequences.

In one embodiment of the invention, such chimeric molecules comprise a fusion of an anti-TAT antibody or TAT polypeptide with a tag polypeptide that provides an epitope to which the anti-tag antibody can selectively bind. Epitope tags are generally located at the amino or carboxyl termini of an anti-TAT antibody or TAT polypeptide. The presence of an anti-TAT antibody or TAT polypeptide in a form with the epitope tag can be detected using an antibody against the tag polypeptide. In addition, the introduction of epitope tags facilitates the purification of anti-TAT antibodies or TAT polypeptides by affinity purification using anti-tag antibodies or other types of affinity matrices that bind to epitope tags. Various tag polypeptides and their respective antibodies are known in the art. Examples include poly-histidine or poly-histidine-glycine tags, flu HA tag polypeptides and antibodies thereof 12CA5 [Field et al., Mol . Cell. Biol . , 8 : 2159-2165 (1988)], c-myc tags and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology , 5 : 3610-3636 (1985)] And herpes simplex virus glycoprotein D (gD) tags and antibodies thereof (Paborsky et al., Protein Engineering , 3 (6): 547-553 (1990)). Other tag polypeptides include Flag-peptide [Hopp et al., BioTechnology , 6 : 1204-1210 (1988)], KT3 epitope peptide [Martin et al., Science , 255 : 192-194 (1992)], alpha-tubulin Epitope peptides [Skinner et al., J. Biol . Chem . , 266 : 15163-15166 (1991)] and the T7 gene 10 protein tag [Lutz-Freyermuth et al., Proc . Natl . Acad . Sci . USA , 87 : 6393-6397 (1990).

In another embodiment, the chimeric molecule may comprise a fusion of an anti-TAT antibody or TAT polypeptide with an immunoglobulin or specific region of an immunoglobulin. For the divalent form of the chimeric molecule (also referred to as "immunoadhesin"), the fusion may be the Fc region of an IgG molecule. This Ig fusion preferably comprises the substitution of a site of one or more variable regions in the Ig molecule with a soluble (deleted or inactivated transmembrane domain) form of an anti-TAT antibody or TAT polypeptide. In one particularly preferred embodiment, the immunoglobulin fusions comprise the hinge, CH ₂ and CH ₃ , or hinge, CH ₁ , CH ₂ and CH ₃ regions of the IgG1 molecule. For a method of producing immunoglobulin fusions, see US Pat. No. 5,428,130, published June 27, 1995.

I. Preparation of Anti-TAT Antibodies and TAT Polypeptides

The following description relates primarily to the production of anti-TAT antibodies and TAT polypeptides by culturing cells transformed or transfected with vectors containing anti-TAT antibodies and TAT polypeptide encoding nucleic acids. Of course, other methods known in the art can be used to prepare anti-TAT antibodies and TAT polypeptides. For example, suitable amino acid sequences or fragments thereof can be prepared by direct peptide synthesis using solid phase techniques. See Stewart et al., Solid-Phase Peptide Synthesis , WH Freeman Co., San Francisco, CA (1969). Merrifield, J. Am. Chem . Soc . , 85 : 2149-2154 (1963)). In vitro protein synthesis can be performed by manual or automated methods. Automated synthesis can be performed using, for example, an Applied Biosystems Peptide Synthesizer (Foster City, CA) according to the manufacturer's instructions. Various fragments of the anti-TAT antibody or TAT polypeptide can be separately synthesized chemically and combined using chemical or enzymatic methods to produce the desired anti-TAT antibody or TAT polypeptide.

1. Isolation of DNA Encoding Anti-TAT Antibody or TAT Polypeptide

DNA encoding an anti-TAT antibody or TAT polypeptide can be obtained from a cDNA library prepared from tissues that are believed to possess and express detectable levels of the anti-TAT antibody or TAT polypeptide mRNA. Thus, DNA of human anti-TAT antibodies or TAT polypeptides can conveniently be obtained from cDNA libraries prepared from human tissue as described in the Examples. Anti-TAT antibodies or TAT polypeptide coding genes can also be obtained from genomic libraries or by known synthetic methods (eg, automated nucleic acid synthesis methods).

The library can be screened using probes designed to identify a gene of interest or a protein encoded by the gene (eg, an oligonucleotide consisting of about 20 to 80 or more bases). Screening of cDNA or genomic libraries using selected probes is performed using standard methods as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Haror Laboratory Press, 1989). can do. Other means for isolating the gene encoding the anti -TAT antibody or TAT polypeptide is to use PCR method [Sambrook et al, supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Haror Laboratory Press, 1995).

Techniques for screening cDNA libraries are well known in the art. Oligonucleotide sequences selected as probes should be sufficiently long and sufficiently clear to minimize false positive results. Oligonucleotides are preferably labeled so that they can be detected upon hybridization to DNA in the library being screened. Labeling methods are known in the art and include the use of radiolabeled, biotinylated or enzymatic labels, such as ³² P-labeled ATP. Hybridization conditions, including moderate stringency and high stringency is provided in the literature [Sambrook et al., Supra.

The sequences identified in the library screening method can be aligned in comparison to other known sequences that can be deposited and obtained in public databases such as GenBank or other proprietary sequence databases. Sequence identity (at the amino acid or nucleotide level) within a defined region of the molecule or across the full length sequence can be determined using methods known in the art and the methods described herein.

Nucleic acid having protein coding sequence is the first reference to detect the precursor, if used to estimate the amino acid sequence disclosed herein and, if necessary [Sambrook et al., Supra usual using the primer extension method, selected cDNA or genomic libraries as described in Can be obtained by screening and processing intermediates of mRNA that are not reverse transcribed into cDNA.

2. Selection and transformation of host cells

Host cells are conventional nutrients that have been transfected or transformed with the expression or cloning vectors described herein for the production of anti-TAT antibodies or TAT polypeptides and modified for promoter induction, transformant selection or amplification of coding genes of the desired sequence. The medium is cultured. Those skilled in the art can select culture conditions such as medium, temperature, pH, etc. without performing unnecessary experiments. In general, principles, protocols, and techniques employed to maximize productivity of cell cultures are described in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., Supra.

Eukaryotic transfection methods and prokaryotic transformation methods such as CaCl ₂ , CaPO ₄ , liposome-mediated methods and electroporation methods are known to those skilled in the art. Depending on the host cell used, transformation is carried out using standard techniques suitable for the cell. Calcium treatment, or electroporation, using the calcium chloride method described in Sambrook et al., Supra, is generally used for prokaryotic cells. Infections with Agrobacterium tumefaciens have been described as described in Shaw et al., Gene , 23 : 315 (1983) and International Publication No. 89/05859 published June 29, 1989. For mammalian cells without cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52: 456-457 (1978) can be used. General characteristics of host system transfection are described in US Pat. No. 4,399,216. Transformation into yeast is generally described by Van Solingen et al., J. Bact., 130: 949 (1977) and Hsiao et al. , Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979), however, other methods of introducing DNA into cells, such as intranuclear microinjection, electroporation, prototypes Fusion of cells with bacterial protoplasts, or polycationics such as polybrene, polyornithine For various techniques for transformation of mammalian cells, see Keown et al., Methods in Enzymology, 185: 527-537 (1990) and Mansour et al., Nature, 336: 348-352 (1988). )].

Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryotic, yeast or higher eukaryotic cells. Suitable prokaryotic cells are sedative bacteria, for example Gram-negative or Gram-positive organisms, for example Enterobacteriaceae , for example E. coli . E. coli , including but not limited to. A variety of teeth. E. coli strains, for example E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537). E. coli strain W3110 (ATCC 27,325) and E. coli. E. coli strain K5 772 (ATCC 53,635) is readily available. Other suitable prokaryotic host cells are Eshcerichia , for example E. coli . E. coli, Enterobacter (Enterobacter), El Winiah (Erwinia), keulrep when Ella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella), for example, Salmonella typhimurium (Salmonella typhimurium), Serratia marcescens (Serratia), for example, For example, Serratia Marsescans Enterobacteriaceae such as marcescans) and Shigella (Shigella) and (Enterobacteriaceae), and Bacillus (Bacillus), for example, rain. B. subtilis and b. Needle piece formate miss (B. licheniformis) (e.g., as set forth in the DD 266,710, published April 12, 1989 Date of rain piece you miss formate (B. licheniformis) 41P), for Pseudomonas (Pseudomonas), for example, blood. Rugi ah include labor (P. aeruginosa), and Streptomyces (Streptomyces). This example is illustrative only and is not limited thereto. Strain W3110 is a particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentation. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 can be modified to result in mutation of the gene encoding the endogenous protein of the host, an example of such a host is E. coli with the complete genotype tonA . E. coli W3110 strain 1A2, complete genotype tonA this with ptr3 . E. coli W3110 strain 9E4, full genotype tonA ptr3 phoA E15 ( argF - lac ) 169 degP ompT this with kan ^r . E. coli W3110 strain 27C7 (ATCC 55,244), complete genotype tonA ptr3 phoA E15 ( argF -lac) 169 degP ompT rbs7 ilvG this with kan ^r . E. coli W3110 strain 37D6, strain 37D6 having a non-kanamycin resistant degP deletion mutation. E. coli W3110 strain 40B4 and E. coli with the Periplasm protease variant disclosed in US Pat. No. 4,946,783, issued Aug. 7, 1990. E. coli strains. Alternatively, in vitro cloning methods such as PCR or other nucleic acid polymerase reactions are suitable.

Full length antibodies, antibody fragments, and antibody fusion proteins are particularly useful when, for example, glycosylation and Fc effector function are not required, for example, the therapeutic antibody is bound to a cytotoxic agent (eg, toxin) and is immunoconjugate. If they are effective in destroying tumor cells, they can be produced by bacteria. The half-life of circulating full-length antibodies is higher. this. Producing in E. coli is a faster, cheaper and more efficient way. Expression of antibody fragments and polypeptides in bacteria is described, for example, in US Pat. No. 5,648,237 (Carter et. Al.), U.S. Patent No. 5, which discloses a translational initiation region (TIR) and signal sequence for optimal translation and secretion. 5,789,199 (Joly et al.) And 5,840,523 (Simmons et al.), The contents of which are incorporated herein by reference. After expression, the antibody is in the form of a soluble fraction. It can be isolated from E. coli cell paste and purified via, for example, a Protein A or G column depending on the isotype. Final purification can be performed similarly to methods for purifying antibodies expressed, for example, in CHO cells.

In addition to prokaryotic cells, eukaryotic microorganisms such as fibrous fungi or yeast are suitable as cloning or expression hosts for anti-TAT antibodies or TAT polypeptide coding vectors. Saccharomyces cerevisiae ) is a commonly used lower eukaryotic host microorganism. Other microorganisms include Schizosaccharomyces pombe [Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383, published May 2, 1985; Kluyveromyces hosts (US Pat. No. 4,943,529; Fleer et al., Bio / Technology, 9: 968-975 (1991)], for example K. et al. K. lactis [MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 737 [1983], k. K. fragilis (ATCC 12,424), K. B. bulgaricus (ATCC 16,045), K. K. wickeramii (ATCC 24,178), K. K. waltii (ATCC 56,500), K. K. drosophilarum [ATCC 36,906; Van den Berg et al., Bio / Technology, 8: 135 (1990). Thermo Toledo Lance (K. thermotolerans) and Kay. Maxianus ( K. marxianus ); Yarrow subtotal (yarrowia) [EP 402 226]; Pichia Pastoris pastoris ) [European Patent 183,070; Sreekrishna et al., J. Basic Microbiol., 28: 265-278 [1988]; Candida (Candida); Trichoderma reesia ) (European Patent 244,234); Neurospora crassa ; Case et al., Proc. Natl. Acad. Sci. USA, 76: 5259-5263 [1979]; Schwanniomyces , for example Schwanniomyces occidentalis ) (European Patent 394,538, published October 31, 1990); And fibrous fungi such as Neurospora , Penicillium , Tolypocladium (International Publication No. 91/00357, published January 10, 1991) and Aspergillus ( Aspergillus ) hosts, for example A. A. nidulans [Ballance et al, Biochem. Biophys. Res. Commun., 112: 284-289 [1983]; Tilburn et al., Gene, 26: 205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984] and A. Needle germanium (A. niger) [Kelly and Hynes , EMBO J., 4: 475-479 [1985]] is included. Methyl auxotrophic yeast are suitable and, a century Cronulla (Hansenula), Candida (Candida), the claw exciter Mosquera (Kloeckera), blood teeth (Pichia), saccharose in my process (Saccharomyces), sat rulrop sheath (Torulopsis) and also torulra ( Rhodotorula ), including but not limited to yeasts that can grow on methanol. For a list of specific species that are examples of these types of yeast, see C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated anti-TAT antibodies or TAT polypeptides are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodopterra Sf9, and cells of plant cells such as cotton, corn, potatoes, soybeans, petunias, tomatoes, and tobacco. Numerous baculovirus strains and variants, and Spodoptera frugiperda ) (Caterpillar), Aedes agetti aegypti ) (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster ) (fruit flies) and Bombyx mori ( Bombyx) mori ) Corresponding acceptable insect host cells derived from the host have been identified. Various virus strains for transfection, for example Autographa Californica califonica ) L-1 variants of NPV and Bm-5 strains of Bombyx mori NPV are available, and such viruses can be used herein as viruses according to the invention, in particular for transfection of Spodoptera pruperfera cells. have.

However, the greatest interest is in vertebrate cells, and propagating vertebrate cells in culture (tissue culture) has become a common process. Examples of useful mammalian host cell lines include monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); Human embryonic kidney cell line [293 cells, or 293 cells subcloned for growth in suspension culture; Graham et al., J. Gen Virol., 36:59, 1977; Baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR [CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA, 77: 4216 (1980); Mouse sertoli cells [TM4, Mather, Biol. Reprod., 23: 243-251 (1980); Monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); Human cervical carcinoma cells (HELA, ATCC CCL 2); Dog kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human liver cells (Hep G2, HB 8065); Mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells [Mather et al., Annals N.Y. Acad. Sci., 383: 44-68 (1982); MRC 5 cells; FS4 cells; And human liver carcinoma cell line (Hep G2).

Host cells are transformed with the above expression or cloning vector for the production of anti-TAT antibodies or TAT polypeptides and then optionally modified to induce promoters, select transformants or amplify genes encoding the desired sequences. In a conventional nutrient medium.

3. Selection and use of replicable vectors

Nucleic acids encoding an anti-TAT antibody or TAT polypeptide (eg cDNA or genomic DNA) can be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors can be easily obtained. For example, the vector may be in the form of a plasmid, cosmid, viral particles or phage. Suitable nucleic acid sequences can be inserted into the vector by various methods. In general, DNA is inserted into suitable restriction enzyme site (s) using techniques known in the art. Vector components generally include, but are not limited to, one or more signal sequences, origins of replication, one or more marker genes, enhancer components, promoters and transcription termination sequences. The preparation of suitable vectors comprising one or more of these components uses standard ligation techniques known to those skilled in the art.

TAT can be produced not only by direct recombination methods, but also as a fusion polypeptide with a heterologous polypeptide, which can be a mature protein or other polypeptide or signal sequence having a specific cleavage site at the N-terminus of the polypeptide. In general, the signal sequence may be a component of the vector or may be part of an anti-TAT antibody or TAT polypeptide coding DNA inserted into the vector. The signal sequence can be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp or thermostable enterotoxin II leader. For secretion in yeast, signal sequences include, for example, a yeast invertase leader, an α factor leader ( Saccharomyces and Kluyveromyces α-factor leader (US Pat. No. 5,010,182)). Or acid phosphatase leader, seed. C. albicans glucoamylase leader (European Patent 362,179 published April 4, 1990) or signal sequence described in International Publication No. 90/13646 published November 15, 1990. In mammalian cell expression, mammalian signal sequences, such as signal sequences from secretory polypeptides of the same or related species, and viral secretion leaders can be used to induce the secretion of proteins.

Both expression and cloning vectors contain nucleic acid sequences that allow the vector to replicate in one or more selected host cells. Such sequences are known for various bacteria, yeasts and viruses. The origin of replication from plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin of replication is suitable for yeast, and various viral origins of replication (SV40, polyoma, adenovirus, VSV or BPV) can be used to detect vectors in mammalian cells. Useful for cloning.

Expression and cloning vectors will typically contain a selection gene, also called a selectable marker. Representative select genes, eg, genes encoding D-alanine racemases for Bacillus, may (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracyclinetetracycline Protein, (b) a protein that compensates for nutrient deficiencies, or (c) a protein that supplies an important nutrient that is not available from the complex medium.

Examples of selectable markers suitable for mammalian cells include those that allow identification of cells that can receive anti-TAT antibodies or TAT polypeptide encoding nucleic acids, for example DHFR or thymidine kinase. When wild type DHFR is used, a suitable host cell is a CHO cell line lacking DHFR activity, see Urlaub et al, Proc. Natl. Acad. Sci. USA, 77: 4216 (1980). Suitable selection genes for use in yeast are the trp1 gene present in yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979); Kingsman et al., Gene, 7: 141 (1979); Tschemper et al., Gene, 10: 157 (1980). The trp1 gene provides a selection marker for variant strains of yeast lacking the ability to grow to tryptophan (eg, ATCC 44076 or PEP4-1) (Jones, Genetics, 85: 12 (1977)).

Expression and cloning vectors generally contain a promoter operably linked to an anti-TAT antibody or TAT polypeptide encoding nucleic acid sequence that induces mRNA synthesis. Promoters recognized by various potential host cells are known. Suitable promoters for use in prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979), alkaline phosphatase, tryptophan (trp) promoter system [Goeddel, Nucleic acid Res., 8: 4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983). In addition, promoters used in bacterial systems will contain a Shine-Dalgarno (S.D.) sequence operably linked to DNA encoding an anti-TAT antibody or TAT polypeptide.

Examples of promoter sequences suitable for use in yeast hosts include 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255: 2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7: 149 (1968); Holland, Biochemistry, 17: 4900 (1978)], for example enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate Promoters for isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosphosphate isomerase, phosphoglucose isomerase and glucokinase.

Other yeast promoters, which are inducible promoters with the additional advantage of transcription controlled by growth conditions, include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes involved in nitrogen metabolism, metallothionein, glycer There is a promoter region for aldehyde-3-phosphate dehydrogenase, and enzymes that act on maltose and galactose use. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

Anti-TAT antibody or TAT polypeptide transcription from a vector in a mammalian host cell may be a virus such as a polyoma virus, Paulpox virus (UK No. 2,211,504 published July 5, 1989), adenovirus (eg , Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and monkey virus 40 (SV40) promoters obtained from genomes, heterologous mammalian promoters such as the actin promoter Or by a promoter suitable for a host cell system, obtained from an immunoglobulin promoter and a heat-shock promoter.

Transcription of the DNA encoding the anti-TAT antibody or TAT polypeptide by higher eukaryotic cells can be increased by inserting an enhancer sequence into the vector. Enhancers are generally cis-acting components of about 10 to 300 bp of DNA, which act on the promoter to increase transcription. Many enhancer sequences are known to be derived from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Typically, however, one will use an enhancer derived from a eukaryotic virus. Examples include the SV40 enhancer on the back of the origin of replication (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the back of the origin of replication, and adenovirus enhancers. The enhancer can be spliced into the vector at the 5 'or 3' position of the anti-TAT antibody or TAT polypeptide coding sequence, but is preferably located at the 5 'site from the promoter.

In addition, expression vectors used in eukaryotic host cells (multinuclear cells derived from yeast, fungi, insects, plants, animals, humans or other multicellular organisms) may comprise sequences necessary for transcription termination and mRNA stabilization. Such sequences are typically obtained from the 5 'and sometimes 3' untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions include nucleotide fragments that are transcribed as polyadenylation fragments in the untranslated portion of the mRNA encoding an anti-TAT antibody or TAT polypeptide.

Other methods, vectors and host cells suitable for application to the synthesis of anti-TAT antibodies or TAT polypeptides in recombinant vertebrate cell culture are described in Genet et al., Nature, 293: 620-625 (1981); Mantei et al., Nature, 281: 40-46 (1979); European Patent Nos. 117,060 and 117,058.

4. Culture of Host Cells

Host cells used to prepare anti-TAT antibodies or TAT polypeptides of the invention can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), minimal essential medium ((MEM), Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) were used to Suitable for cultivation See also, Ham et al., Meth. Enz., 58: 44 (1979), Barnes et al., Anal. Biochem., 102: 255 (1980), US Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; Or 5,122,469; WO 90/03430; WO 87/00195; Or any of the mediums described in US Patent No. 30,985 can be used as the culture medium for the host cells. Any of these media may be used as needed, including hormones and / or other growth factors (eg, insulin, transferrin or epidermal growth factor), salts (eg, sodium chloride, calcium, magnesium and phosphate), buffers ( For example, HEPES), nucleotides (eg adenosine and thymidine), antibiotics (eg gentamicin® drugs), trace elements (typically inorganic compounds present in final concentrations in the micromolar range) And glucose or equivalent energy sources. Any other necessary supplements may also be included at appropriate concentrations known in the art. Culture conditions such as temperature, pH, etc. are conditions that are already in use with a host cell selected for expression, which will be apparent to those skilled in the art.

5. Detection of Gene Amplification and Expression

Gene amplification and / or expression may be used, for example, in conventional Southern blotting, Northern blotting to quantify transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980)], dot blotting (DNA analysis) or in situ hybridization with appropriately labeled probes based on the sequences provided herein. Alternatively, antibodies can be used that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. In other words, an antibody can be labeled and the double chain bound to the surface to perform an assay that can detect the presence of the antibody bound to the double chain when the double chain is formed on the surface.

Alternatively, gene expression can be measured by immunological methods such as immunohistochemical staining of cells or tissue fragments and analysis of cell culture or body fluids for direct quantification of expression of gene products. Antibodies useful for immunohistochemical staining and / or analysis of sample fluids may be monoclonal or polyclonal antibodies, which may be prepared in any mammal. Conveniently, antibodies can be made to native sequence TAT polypeptides, synthetic peptides based on the DNA sequences provided herein, or to foreign sequences that are fused to TAT DNA and that encode specific antibody epitopes.

6. Purification of Anti-TAT Antibodies and TAT Polypeptides

Forms of anti-TAT antibodies and TAT polypeptides can be recovered from culture medium or host cell lysates. When bound to the cell membrane, it may be released from the cell membrane using a suitable detergent solution (eg Triton-X 100) or by cleavage of the enzyme. Cells used for expression of anti-TAT antibodies and TAT polypeptides can be milled by various physical or chemical means such as freeze-thaw cycles, sonication, mechanical milling or cell lysing agents.

It may be desirable to purify anti-TAT antibodies and TAT polypeptides from recombinant cellular proteins or polypeptides. Examples of suitable purification methods include fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica or cation exchange resins such as chromatography on DEAE, chromatographic focusing, SDS-PAGE, ammonium sulfate precipitation, Sephadex Gel filtration using G-75, Protein A Sepharose column to remove contaminants such as IgG, and metal chelating column to bind epitope tag forms of anti-TAT antibodies and TAT polypeptides. Various protein purification methods can be used and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step (s) selected will depend, for example, on the production method used and the nature of the particular anti-TAT antibody or TAT polypeptide produced.

When using recombinant technology, antibodies can be produced in the periplasm of cells or secreted directly into the medium. Once the antibody is produced in the cell, as a first step, centrifugation or ultrafiltration is performed to remove particulate debris, which is a host cell or degradation fragment of the host. this. See Carter et al., Bio / Technology 10: 163-167 (1992) for methods for isolating antibodies secreted into the periplasmic space of E. coli. In brief, the cell paste is thawed over about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonylfluoride (PMSF). Cell debris is removed by centrifugation. When the antibody is secreted into the medium, the supernatant obtained from this expression system is first concentrated using a commercial protein concentration filter such as Amicon or Millipore Pelicon Ultrafiltration. Protease inhibitors such as PMSF can be included in any of these steps to inhibit proteolysis and antibiotics can also be included to prevent the growth of foreign contaminants.

Antibody compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of Protein A as an affinity ligand of Protein A depends on the species and isotype of the immunoglobulin Fc domain present in the antibody. Protein A can be used to purify human γ1, γ2 or γ4 heavy chain-based antibodies [Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J 5: 1567-1575 (1986)). The matrix to which the affinity ligand will be attached is mostly agarose, but other matrices are available. Mechanically stable matrices such as pore-adjusted glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than agarose. If the antibody comprises a C _H 3 domain, Bakerbond ABX® resin (JT Baker; Phillipsburg, NJ) is useful for purification. Fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE®, or chromatography on an anion or cation exchange resin (eg polyaspartic acid column), depending on the antibody to be recovered. Other protein purification techniques such as, chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation can also be used.

After any preliminary purification step (s), using an elution buffer at a pH of about 2.5 to 4.5 and a low salt concentration (eg, about 0-0.25 M salt) for the mixture containing the desired antibody and contaminants, Low pH hydrophobic interaction chromatography can be performed.

J. Pharmaceutical Formulations

Therapeutic preparations of anti-TAT antibodies, TAT binding oligopeptides, TAT binding organic molecules and / or TAT polypeptides used in accordance with the present invention may be prepared by any pharmaceutical agent comprising an antibody, polypeptide, oligopeptide or organic molecule exhibiting the desired purity. Acceptable carriers, excipients or stabilizers [Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), for storage in the form of a lyophilized formulation or aqueous solution. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as acetates, tris, phosphates, citrate and other organic acids; Antioxidants including ascorbic acid and methionine; Preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol Cyclohexanol; 3-pentanol; and m-cresol); Low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; Monosaccharides, disaccharides and other carbohydrates, including glucose, mannose, or dextrins; Chelating agents such as EDTA; Reinforcing agents such as trehalose and sodium chloride; Sugars such as sucrose, mannitol, trehalose or sorbitol; Surfactants such as polysorbate; Salt-forming counterions such as sodium; Metal complexes such as Zn-protein complexes; And / or nonionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG). Preferably the formulation comprises an antibody at a concentration of 5-200 mg / ml, preferably at a concentration of 10-100 mg / ml.

The formulations herein may contain, if necessary, one or more active compounds for the particular condition being treated, preferably compounds which exhibit complementary activity that do not adversely affect each other. For example, in addition to an anti-TAT antibody, a TAT binding oligopeptide or a TAT binding organic molecule, other antibodies, such as a second anti-TAT antibody that binds to another epitope on a TAT polypeptide, or affect the growth of a particular cancer It may be desirable to include in the formulation an antibody against some other target, such as a growth factor. Alternatively or additionally, the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and / or cardioprotectant. Such molecules are suitably present together in an amount effective for the purpose intended.

The active ingredient may also be placed in microcapsules prepared by coacervation techniques or by interfacial polymerization reactions (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively). Colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nano-particles and nano-capsules) or macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained release formulations may be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactide (US Pat. No. 3,773,919), L- Copolymers of glutamic acid with γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT® (lactic acid-glycolic acid copolymer and leuprolide acetate Injectable microspheres), and poly-D-(-)-3-hydroxybutyric acid.

The formulation to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

K. Diagnosis and Treatment with Anti-TAT Antibodies, TAT Binding Oligopeptides, and TAT Binding Organic Molecules

Various diagnostic assays are available for confirming TAT expression in cancer. In one embodiment, overexpression of the TAT polypeptide can be analyzed by immunohistochemistry (IHC). Paraffin embedded tissue sections from tumor biopsies can be analyzed by IHC and the following TAT protein staining intensity criteria can be recorded:

Score 0

No staining is observed or membrane staining is observed in less than 10% of tumor cells.

Score 1+

Faintly recognizable membrane staining is detected in at least 10% of tumor cells. These cells are stained only in some of their membranes.

Score 2+

Weak to moderate levels of membrane staining are observed in more than 10% of tumor cells.

Score 3+

Medium to strong levels of membrane staining are observed in more than 10% of tumor cells.

Tumors that score 0 or 1+ for the evaluation of TAT polypeptide expression may be characterized by not overexpressing TAT, while tumors that score 2+ or 3+ may be characterized by overexpression of TAT.

Alternatively or additionally, FISH analysis, for example INFORM® (Ventana, Arizona) or PATHVISION® (Vysis, Illinois), is performed on tumor tissues fixed in formalin and embedded in paraffin. The extent of TAT overexpression in the tumor, if any, can be measured.

TAT overexpression or amplification involves the administration of a molecule (eg, an antibody, oligopeptide or organic molecule) that binds to a molecule to be detected and is tagged with a detectable label (eg, a radioisotope or fluorescent label) and the label is concentrated. Measurements can be made using in vivo diagnostic assays by external scanning of the patient to locate the location.

As described above, anti-TAT antibodies, oligopeptides or organic molecules of the invention may be used in a variety of non-therapeutic applications. Anti-TAT antibodies, oligopeptides or organic molecules of the invention may be useful for the diagnosis and staging (eg, radioimaging) of TAT-expressing cancer. Antibodies, oligopeptides or organic molecules of the invention may be mixed in the purification of other cells by purifying or immunoprecipitating the TAT polypeptide from cells to detect and quantify TAT-polypeptides in vitro, for example, by ELISA or Western blot. It is also useful for killing and eliminating TAT-expressing cells in one step from the population of cells.

Currently, depending on the stage of the cancer, the cancer is treated by one of surgery, radiation therapy and chemotherapy to remove cancer tissue or a combination of the above therapies. Anti-TAT antibodies, oligopeptides or organic molecular therapies may be particularly desirable in older patients who do not tolerate the toxicity and side effects of chemotherapy well for metastatic disease in which the use of radiation therapy is limited. Tumor targeting anti-TAT antibodies, oligopeptides or organic molecules of the invention are useful for alleviating TAT-expressing cancers such as those diseases when they are initially diagnosed or during relapse. When used in therapy, anti-TAT antibodies, oligopeptides or organic molecules may be used alone or in combination with a compound labeled with, for example, hormones, antiangiogenic agents or radioisotopes, or in particular, It may be used in combination with surgery, cryotherapy and / or radiotherapy. Anti-TAT antibodies, oligopeptides or organic molecules can be administered in conjunction with other forms of conventional therapy or administered sequentially before and after conventional treatment regimens. Chemotherapeutic drugs such as taxoter (doxtaxel), taxol (paclitaxel), esturamustine and mitoxantrone are used for the treatment of cancer, especially for very dangerous patients. In the methods of the invention for use in the treatment or alleviation of cancer, a cancer patient may be administered an anti-TAT antibody, oligopeptide or organic molecule with one or more of the above chemotherapeutic agents. In particular, concomitant therapies with paclitaxel and modified derivatives (see for example European Patent No. 0600517) are also contemplated. The anti-TAT antibody, oligopeptide or organic molecule will be administered with a therapeutically effective amount of chemotherapeutic agent. In another embodiment, the anti-TAT antibody, oligopeptide or organic molecule is administered in combination with chemotherapy to elevate the activity and efficacy of a chemotherapeutic agent such as paclitaxel. Physician tabletop manuals (PDRs) disclose dosages of these chemotherapeutic agents for use in the treatment of various cancers. The dosage and dosage of the chemotherapeutic agent that produces a therapeutic effect depends on the particular cancer to be treated, the severity of the cancer, and other factors known to those skilled in the art and can be determined by the physician.

In one specific embodiment, an immunoconjugate comprising an anti-TAT antibody, oligopeptide or organic molecule combined with a cytotoxic agent is administered to the patient. Preferably, when the immunoconjugate bound to the TAT protein is introduced into the cell, the therapeutic efficacy of the immunoconjugate against death of cancer cells bound to the immunoconjugate is increased. In a preferred embodiment, the cytotoxic agent targets or interferes with the nucleic acid of the cancer cell. Examples of such cytotoxic agents are described above and include maytansinoids, calicheamicins, ribonucleases and DNA endonucleases.

Anti-TAT antibodies, oligopeptides or organic molecules or toxin conjugates thereof can be prepared by known methods such as bolus, or by continuous irrigation over a period of time, intravenous, intramuscular, intraperitoneal, intrathecal, spinal, subcutaneous, joint It is administered to human patients by intraoral, intracapsular, intravesical, oral, topical or inhalation routes. Intravenous or subcutaneous administration of the antibody, oligopeptide or organic molecule is preferred.

Other therapeutic methods can be combined with the administration of anti-TAT antibodies, oligopeptides or organic molecules. Concomitant administration methods include administration together using separate or single pharmaceutical preparations, and successive administrations in any order, where two (or all) active ingredients simultaneously share their biological activity. It is desirable to have a time to exercise. Preferably such concurrent dosing regimens exert a synergistic therapeutic effect.

Administration of the anti-TAT antibody (s), oligopeptides or organic molecules may preferably be combined with administration of the antibody to another tumor associated antigen associated with the particular cancer.

In one embodiment, the antibody therapy of the present invention involves the concurrent administration of an anti-TAT antibody (or antibodies), oligopeptide or organic molecule with one or more chemotherapeutic or growth inhibitory agents, including Included are methods of simultaneous administration of a cocktail of therapies. Chemotherapeutic agents include esthramustine phosphate, prednismustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and hydroxyureataxanes (e.g., paclitaxel and docetaxel) and ( Or) anthracycline antibiotics. Preparation and dosing schedules for such chemotherapeutic agents may be used according to the manufacturer's instructions or as determined experimentally by one skilled in the art. Preparation and dosing schedules for such chemotherapy are described in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, MD (1992).

Antibodies, oligopeptides or organic molecules include anti-estrogen compounds such as anti-hormone compounds such as tamoxifen; Anti-progesterone such as onapristone (see EP 616 812); Or anti-androgen compounds such as flutamide (the anti-hormone compounds are used in known dosages for them). If the cancer to be treated is an androgen-independent cancer, the patient may be previously treated with anti-androgen therapy, and after the cancer has been androgen-independent, an anti-TAT antibody, oligopeptide or organic molecule (and optionally described herein) Other agents) may be administered to the patient.

Often, it may be beneficial to administer a cardioprotectant (to prevent or alleviate myocardial insufficiency associated with the therapy) or one or more cytokines to the patient. In addition to the above treatments, the patient may be surgically removed to remove cancer cells and / or treated with radiation therapy prior to, concurrent with or after antibody, oligopeptide or organic molecular therapy. Suitable dosages for any of the agents administered together are those currently in use and may be lowered due to the synergy of the agents with anti-TAT antibodies, oligopeptides or organic molecules.

To prevent or treat a disease, the physician will select the dosage and mode of administration according to known criteria. Appropriate dosages of antibodies, oligopeptides or organic molecules may be determined by the type of disease to be treated as defined above, whether the antibody, oligopeptide or organic molecule is administered for prophylactic or therapeutic purposes, Prior treatment regimens, the patient's clinical history and responsiveness to antibodies, oligopeptides or organic molecules, and the physician's discretion will be determined. Antibodies, oligopeptides or organic molecules are suitable for single administration to a patient or throughout a series of treatments. Preferably, the antibody, oligopeptide or organic molecule is administered by intravenous irrigation or subcutaneous injection. Depending on the type and severity of the disease in question, the initial candidate dose for administration to the patient may be from about 1 μg to about 50 mg of antibody per kg of body weight (eg, from about 0.1 to 15, regardless of one or more individual or continuous irrigation). Mg / dose). Dosing may include administering an initial loading dose of about 4 mg / kg of anti-TAT antibody followed by weekly administration of a maintenance dose of about 2 mg / kg of anti-TAT antibody. However, other dosage methods are also available. Typical daily dosages will range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. In the case of repeated administration over several days or more depending on the symptoms, treatment is continued until the symptoms of the disease are suppressed as desired. The progress of this therapy is easily monitored by conventional techniques and assays based on criteria known to the physician or person skilled in the art.

Apart from administering the antibody protein to the patient, the present application contemplates administering the antibody by gene therapy. Nucleic acid encoding an antibody is included in the expression "administering a therapeutically effective amount of an antibody." See, for example, WO 96/07321 (published on March 14, 1996) concerning the use of gene therapy to generate intracellular antibodies.

There are two main methods (in vivo and ex vivo) for injecting nucleic acid (optionally contained in a vector) into a patient's cell. For in vivo delivery, the nucleic acid is usually injected directly into the patient at the site where the antibody is desired. For ex vivo treatment, the cells of the patient are taken out and then the nucleic acid is introduced into these isolated cells and such modified cells are administered directly to the patient, or encapsulated in, for example, a porous membrane implanted into the patient. In US Pat. No. 4,892,538; And 5,283,187. There are a variety of techniques available for introducing nucleic acids into living cells. Such techniques vary depending on whether they are delivered into cells cultured in vitro or in vivo into cells of the intended host. Suitable techniques for delivering nucleic acids to mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. A vector commonly used for ex vivo delivery of such genes is a retroviral vector.

Currently preferred in vivo nucleic acid delivery techniques include viral vectors (eg, adenoviruses, herpes simplex I virus, or adeno-associated viruses) and lipid-based systems (lipids useful for lipid mediated delivery of genes include, for example, DOTMA, DOPE, and Transfection with DC-Chol). For details on currently known gene marking and gene therapy protocols, see Anderson et al., Science 256: 808-813 (1992); See WO 93/25673 and references cited in these patents.

Anti-TAT antibodies of the invention may be in various forms included in the definition of "antibody" herein. Thus, antibodies include full length or complete forms of antibodies, antibody fragments, native sequence antibodies or amino acid variants, humanized antibodies, chimeric or fusion antibodies, immunoconjugates, and functional fragments thereof. In fusion antibodies, the antibody sequence is linked to a heterologous polypeptide sequence. Antibodies can be modified in the Fc region to provide the desired effector function. As described in more detail in the following paragraphs, an exposed antibody bound to the cell surface and comprising an appropriate Fc region is, for example, through antibody dependent cell mediated cytotoxicity (ADCC), or by a complement dependent cytotoxic mechanism. Cytotoxicity can be induced through the circulation of complement or other mechanisms. Alternatively, if it is desired to eliminate or reduce effector function to minimize side effects or therapeutic complications, certain other Fc regions can be used.

In one embodiment, the antibodies compete for binding to or substantially binding the same epitope as the antibodies of the invention. Antibodies that exhibit the biological properties of the anti-TAT antibodies of the invention include, in particular, those that include properties that specifically bind to tumors in vivo, and those that inhibit cell proliferation or are toxic to cells.

The method of producing the antibody is described in detail below.

Anti-TAT antibodies, oligopeptides, and organic molecules of the invention are useful for treating TAT-expressing cancers in mammals or alleviating one or more symptoms of such cancers. Such cancers include prostate cancer, urethral cancer, lung cancer, breast cancer, colon cancer and ovarian cancer, more specifically prostate adenocarcinoma, renal cell carcinoma, colorectal adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma and pleural mesothelioma. Cancers include any of the metastatic cancers described above. Antibodies, oligopeptides and organic molecules can bind to at least some of the cancer cells that express a TAT polypeptide in a mammal. In a preferred embodiment, the antibodies, oligopeptides and organic molecules are effective for destroying or killing TAT-expressing tumor cells or inhibiting the growth of such tumor cells in vitro or in vivo when bound to a TAT polypeptide on a cell. Such antibodies include exposed anti-TAT antibodies (no substance bound). Exposed antibodies that exhibit cytotoxic or cell growth inhibitory properties can also be combined with cytotoxic agents that make the antibody much more potent in destroying tumor cells. For example, cytotoxicity can be imparted to anti-TAT antibodies by combining the antibody with a cytotoxic agent to form the following immunoconjugates. Cytotoxic agents or cell growth inhibitors are preferably small molecules. Preferred are toxins such as calicheamicin or maytansinoids and their homologues or derivatives.

The present invention provides a composition comprising an anti-TAT antibody, oligopeptide or organic molecule of the invention and a carrier. To treat cancer, the composition may be administered to a human in need thereof, wherein the composition may comprise one or more anti-TAT antibodies or exposed antibodies present as immunoconjugates. In further embodiments, the compositions of the present invention may comprise these antibodies, oligopeptides or organic molecules with other therapeutic agents, such as cytotoxic agents or growth inhibitors, including chemotherapeutic agents. The invention also provides formulations comprising an anti-TAT antibody, oligopeptide or organic molecule of the invention and a carrier. In one embodiment, the formulation is a therapeutic formulation comprising a pharmaceutically acceptable carrier.

Another aspect of the invention is an isolated nucleic acid encoding an internalizing anti-TAT antibody. Also included are nucleic acids encoding both H or L chains and in particular hypervariable region residues, nucleic acids encoding native sequence antibodies, and nucleic acids encoding variants, variants and humanized antibodies of these antibodies.

The invention also provides methods useful for treating or alleviating one or more symptoms of TAT polypeptide expressing cancer in a mammal, comprising administering to the mammal a therapeutically effective amount of an anti-TAT antibody, oligopeptide or organic molecule. do. Antibodies, oligopeptides or organic molecular therapeutic compositions may be administered for a short period (acute) or for a long time or intermittently, as directed by a physician. The present invention also provides a method of inhibiting the growth of cells expressing TAT polypeptide and killing the cells.

The invention also provides kits and articles comprising one or more anti-TAT antibodies, oligopeptides or organic molecules. Kits containing anti-TAT antibodies are used to purify or immunoprecipitate TAT polypeptides from cells, for example, to analyze killing of TAT cells. For example, for isolation and purification of TAT, the kit may contain an anti-TAT antibody, oligopeptide or organic molecule coupled to the beads (eg, sepharose beads). Kits containing antibodies, oligopeptides or organic molecules for detecting and quantifying TAT by ELISA or Western blot in vitro can be provided. Antibodies, oligopeptides or organic molecules useful for detection may be provided with a label such as a fluorescent label or radiolabel.

L. Products and Kits

In another embodiment of the present invention, a product containing a substance useful for the treatment of anti-TAT expressing cancer is provided. Such products include a container and a label or package insert attached to or attached to the container. Suitable containers include, for example, bottles, vials, syringes and the like. Such containers can be formed from various materials such as glass or plastic. The container holds a composition that is effective for treating the symptoms of cancer and may have a sterile outlet (eg, such a container is an intravenous solvent bag or vial with a stopper that can be penetrated with a hypodermic needle) Can be). At least one active agent in such a composition is an anti-TAT antibody, oligopeptide or organic molecule of the invention. The label or package insert states that the composition is used for the treatment of cancer. The label or package insert will also include instructions for administering the antibody, oligopeptide or organic molecular composition to the patient suffering from the cancer. In addition, the product may further comprise a second container comprising a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. The product may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

Kits are also provided that are useful for a variety of purposes, such as for assaying TAT expressing cell death, for purifying or immunoprecipitating TAT polypeptide from cells. For isolation and purification of TAT polypeptides, the kit may contain an anti-TAT antibody, oligopeptide or organic molecule coupled to the beads (eg, sepharose beads). In vitro, a kit containing an antibody, oligopeptide or organic molecule for detecting and quantifying a TAT polypeptide, for example by ELISA or Western blot, can be provided. As with the product, the kit includes a container and a label or package insert on or attached to the container. The container contains a composition comprising at least one anti-TAT antibody, oligopeptide or organic molecule of the invention. For example, other containers containing diluents and buffers, control antibodies may also be included. The label or package insert can provide a description of the composition as well as a description for use in the desired in vitro or diagnostic.

Uses of M. TAT polypeptides and TAT polypeptide encoding nucleic acids

Nucleotide sequences encoding TAT polypeptides (or complements thereof) have a variety of uses in the field of molecular biology, including their use as hybridization probes, in chromosome and gene mapping and in the preparation of antisense RNA and DNA probes. In addition, TAT encoding nucleic acids will also be useful for the production of TAT polypeptides by the recombinant techniques described herein, which may be used, for example, in the production of anti-TAT antibodies described herein.

The full length native sequence TAT gene or fragment thereof may be used to isolate a full length TAT cDNA or other cDNA (eg, a gene encoding a TAT from a naturally occurring variant or other species of TAT) having the desired sequence identity with the native TAT sequence disclosed herein. Can be used as a hybridization probe for the cDNA library. Optionally, the probe will be about 20 to about 50 bases in length. Hybridization probes can be derived from at least a portion of a new region of the full-length native nucleotide sequence, where this region can be determined without unnecessary experimentation, or from a genomic sequence comprising a promoter, enhancer component and intron of the native sequence TAT. For example, the screening method will include isolating the coding region of the TAT gene using known DNA sequences to synthesize a selected probe of about 40 bases. Hybridization probes can be labeled with a variety of labels, including radioactive nucleotides such as ³² P or ³⁵ S or enzyme labels such as alkaline phosphatase coupled to the probe via an avidin / biotin coupling system. Labeled probes having sequences complementary to the TAT gene of the invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine the members of the library that the probe hybridizes to. Hybridization techniques are described in more detail in the Examples below. Any EST sequence disclosed herein can be similarly used as a probe using the methods described herein.

Other useful fragments of TAT-encoding nucleic acids include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (RNA or DNA) capable of binding to a target TAT mRNA (sense) or TAT DNA (antisense) sequence. Antisense or sense oligonucleotides according to the present invention comprise fragments of coding regions of TAT DNA. This fragment generally comprises at least about 14 nucleotides, preferably about 14 to 30 nucleotides. The ability to induce antisense or sense oligonucleotides based on cDNA sequences encoding the protein of interest is described, for example, in Stein and Cohen ( Cancer Res. 48: 2659, 1988) and van der Krol et al. ( BioTechniques 6: 958, 1988).

Combination of the antisense or sense oligonucleotide with the target nucleic acid sequence forms a double strand that blocks the transcription or translation of the target sequence by one of several or other methods, including enhanced digestion of the double strand, early termination of transcription or translation. . Such a method is included in the present invention. Thus, antisense oligonucleotides can be used to block the expression of TAT proteins, which can play a role in inducing cancer in mammals. In addition, antisense or sense oligonucleotides include oligonucleotides having modified sugar-phosphodiester backbones (or other sugar bonds, eg, sugar bonds disclosed in WO 91/06629), wherein such sugar bonds are internal nucleas There is resistance to the agent. Such oligonucleotides with resistant sugar bonds are stable in vivo (ie, resistant to enzymatic degradation) but retain sequence specificity capable of binding to the target nucleotide sequence.

Other examples of sense or antisense oligonucleotides include organic residues, such as those disclosed in WO 90/10048 and other residues that increase the affinity of the oligonucleotide for the target nucleic acid sequence, such as poly- (L-lysine). Covalently linked oligonucleotides are included. In addition, an insertion agent, such as ellipsine, and an alkylating agent or metal complex can be linked to the sense or antisense oligonucleotide to change the binding specificity of the antisense or sense oligonucleotide to the target nucleotide sequence.

Cells containing the target nucleic acid sequence, for example, by using any gene transfer method, including CaPO ₄ -mediated DNA transfection, electroporation, or by using a gene transfer vector such as Epstein-Barr virus. Antisense or sense oligonucleotides can be introduced into. In one preferred method, antisense or sense oligonucleotides are inserted into a suitable retroviral vector. The cell comprising the target nucleic acid sequence is contacted with the recombinant retroviral vector in vivo or ex vivo. Suitable retroviral vectors include vectors derived from double copy vectors referred to as murine retrovirus M-MuLV, N2 (retrovirus derived from M-MuLV), or DCT5A, DCT5B and DCT5C (see WO 90/13641). Included but not limited to.

In addition, as disclosed in WO 91/04753, it is possible to introduce a sense or antisense oligonucleotide into a cell comprising a target nucleotide sequence by forming a conjugate with a ligand binding molecule. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the binding of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind its corresponding molecule or receptor, nor does it block intracellular entry of the sense or antisense oligonucleotides or conjugate forms thereof.

Alternatively, as disclosed in WO 90/10448, one can introduce a sense or antisense oligonucleotide into a cell comprising a target nucleic acid sequence by forming an oligonucleotide-lipid conjugate. Such sense or antisense oligonucleotide-lipid conjugates are preferably degraded by endogenous lipases in the cell.

Antisense or sense RNA or DNA molecules generally have about 5 or more nucleotides, or about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 , 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220 , 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 , 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720 , 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940 , 950, 960, 970, 980, 990, or 1000 or more in length, wherein the term "about" means the length of a reference nucleotide sequence plus or minus 10% of its length. .

The probe can also be used in PCR techniques to generate a group of sequences for the identification of closely related TAT coding sequences.

In addition, nucleotide sequences encoding TAT can be used to prepare hybridization probes for mapping of genes encoding TAT and for genetic analysis of individuals with genetic disorders. Nucleotide sequences provided herein can be mapped to specific regions of chromosomes and chromosomes using known techniques such as in situ hybridization, association analysis for known chromosomal markers, and hybridization screening using libraries.

If the coding sequence for a TAT encodes a protein that binds to another protein (eg, where TAT is a receptor), the TAT can be used for analysis to identify other proteins or molecules involved in this binding interaction. . By this method, inhibitors of receptor / ligand binding interactions can also be identified. In addition, proteins involved in the binding interaction can be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Receptor TAT can also be used to isolate similar ligands. Screening assays can be designed to find lead compounds that mimic the biological activity of TAT or receptors on TAT. Such screening assays include high throughput screening assays for chemical libraries, and will be particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds. Assays can be performed in a variety of formats including protein-protein binding assays, biochemical screening assays, immunoassays, and cell based assays that are characterized in the art.

Nucleic acids encoding TAT or variants thereof may also be used to generate transgenic or “knock out” animals useful for the development and screening of useful therapeutics. Transgenic animals (eg, mice or rats) are animals that have cells that contain transgenes, which are introduced into the animal or its ancestors during prenatal, eg embryonic stages. do. The transgene is DNA inserted into the genome of a cell from which a transgenic animal is generated. In one embodiment, a cDNA encoding a TAT can be used to clone genomic DNA encoding the TAT according to established techniques and to use the genomic sequence to transform a cell comprising a cell expressing the DNA encoding the TAT. Animals can be produced. In particular, methods for producing transgenic animals such as mice or rats are routine methods in the art and are described, for example, in US Pat. Nos. 4,736,866 and 4,870,009. Typically, specific cells are targeted for the introduction of TAT transgenes with tissue specific enhancers. In an embryonic stage, a transgenic animal comprising a copy of a transgene encoding a TAT introduced into an animal's germ line can be used to investigate the effect of increasing expression of the DNA encoding the TAT. Such animals can be used, for example, as test animals for reagents that are thought to protect from pathological symptoms associated with overexpression of TAT. In accordance with this aspect of the invention, treatment of the animal with the reagent results in a lower incidence of pathological symptoms than untreated animals carrying the transgene, indicating a potential therapeutic intervention in the pathological condition.

Alternatively, a defect or modification encoding TAT as a result of homologous recombination between an endogenous gene encoding TAT using a non-human homologue of TAT and a modified genomic DNA encoding TAT introduced into embryonic stem cells of this animal. It can be used to make TAT "knock out" animals with genes. For example, cDNA encoding TAT can be used to clone genomic DNA encoding TAT according to established techniques. Portions of genomic DNA encoding TAT may be deleted or substituted with other genes, such as genes encoding selective markers that can be used to monitor insertion. In general, thousands of bases of unmodified flanking DNA (both at the 5 ′ and 3 ′ ends) are included in a vector (eg, for homologous recombinant vectors, see Thomas and Capecchi, Cell , 51: 503). (1987). This vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA and homologous DNA are homologously recombined are selected (see, eg, Li et al., Cell , 69). : 915 (1992). Selected cells are then injected into blastocysts of animals (eg, mice or rats) to form aggregated chimeras (see, eg, Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ Robertson, ed. , Oxford, 1987), pp. 113-152). Chimeric embryos were then transplanted into suitable fertility surrogate animals to generate "knock out" animals. Progeny carrying homologous recombinant DNA in germ cells can be identified using standard techniques and used to breed animals in which all cells of the animal contain homologous recombinant DNA. Knockout animals are characterized, for example, by the ability to defend against certain pathological symptoms and the development of pathological symptoms due to the absence of TAT polypeptide.

Nucleic acids encoding TAT polypeptides can also be used for gene therapy. When used in gene therapy, genes are introduced into cells to achieve in vivo synthesis of therapeutically effective gene products, for example to replace defective genes. “Gene therapy” includes both traditional gene therapy where the effect is sustained by a single treatment and administration of gene therapy agents including single or repeated administration of therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic agents to block the expression of certain genes in vivo. Since the uptake of short antisense oligonucleotides through cell membranes is limited, it has already been found that, despite their low intracellular concentrations, they can act as inhibitors when introduced into cells [Zamecnik et al., Proc . Natl . Acad ., Sci . USA 83, 4143-4146 (1986)]. Such oligonucleotides can enhance the uptake of oligonucleotides by modification, for example by replacing their negatively charged phosphodiester groups with non-charged groups.

There are a variety of techniques that can be used to introduce nucleic acids into living cells. This technique depends on whether the nucleic acid is delivered to cells cultured in vitro or to cells of the desired host in vivo. Suitable techniques for delivering nucleic acids to mammalian cells in vitro include liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. Currently preferred gene transfer techniques in vivo include transfection with viral (usually retroviral) vectors and viral coat protein-liposomal mediated transfection [Dzau et al., Trends in Biotechnology 11, 205-210 ( 1993). In some cases, it is desirable to provide the nucleic acid source with an active agent that targets the target cell, such as cell surface membrane proteins or antibodies specific for the target cell, ligands for receptors on the target cell, and the like. When liposomes are used, proteins that bind to cell surface membrane proteins involved in endocytosis can be used for targeting and / or promote uptake, such examples of which show affinity for a particular cell type. Capsid proteins or fragments thereof, antibodies to proteins that undergo internalization in circulation, proteins that target intracellular locations and enhance intracellular half-life. Receptor mediated endocytosis techniques are described, for example, in Wu et al., J. Biol . Chem . 262, 4429-4432 (1987), and Wagner et al., Proc. Natl . Acad . Sci . USA 87, 3410-3414 (1990). For a review of gene marking and gene therapy protocols, see Anderson et al., Science 256, 808-813 (1992).

Nucleic acid molecules or fragments thereof encoding the TAT polypeptides disclosed herein are useful for the identification of chromosomes. In this regard, there is a continuing need to identify new chromosomal markers because there are currently relatively few chromosomal marking reagents available based on actual sequence data. Each of the TAT nucleic acid molecules of the invention can be used as a chromosome marker.

The TAT polypeptides and nucleic acid molecules of the invention can also be used for tissue typing, wherein the TAT polypeptides of the invention are diseased in one tissue, preferably in diseased tissue, compared to normal cells of the same tissue type compared to other tissues. It can be expressed specifically in TAT nucleic acid molecules will be used to generate probes for PCR, Northern analysis, Southern analysis and Western analysis.

The present invention includes methods for identifying compounds (agonists) that mimic TAT polypeptides and for screening compounds (antagonists) to interfere with the effects of TAT polypeptides. Screening assays for antagonist drug candidates can be designed to bind or conjugate to a TAT polypeptide encoded by a gene identified herein, or to interfere with the interaction of the encoded polypeptide with other intracellular proteins, eg, from cells. Compounds that inhibit the expression of TAT polypeptides were identified. Such screening assays include assays that can be highly screened for chemical libraries, which would be particularly suitable for identifying small molecule drug candidates.

Such assays can be performed in a variety of formats including protein-protein binding assays, biochemical screening assays, immunoassays and cell-based assays characterized in the art.

Common to all assays for antagonists is that they require contact of the drug candidate with the TAT polypeptide encoded by the nucleic acid identified herein for conditions and time sufficient to interact.

In binding assays, the interaction is binding, and the conjugate formed can be isolated or detected in the reaction mixture. In certain embodiments, a TAT polypeptide or drug candidate encoded by a gene identified herein is immobilized on a solid, eg, microtiter plate, by covalent or non-covalent attachment. Non-covalent attachment is generally accomplished by coating the solid surface with a TAT polypeptide solution and drying. Alternatively, immobilized antibodies, such as monoclonal antibodies specific for immobilized TAT polypeptides, can be used to anchor them to a solid surface. This analysis is performed by adding an unfixed component that can be labeled with a detectable label to a coding surface comprising a fixed component, for example an anchored component. When the reaction is finished, unreacted components are removed, for example by washing, and the binder anchored to the solid surface is detected. When components that are not originally immobilized carry a detectable label, detection of the label immobilized on the surface indicates that a conjugate formation reaction has occurred. If a component that is not originally immobilized does not carry a label, for example, labeled antibodies that specifically bind to the immobilized conjugate can be used to detect the binder formation reaction.

If a candidate compound interacts with, but does not bind to, a particular TAT polypeptide encoded by a gene identified herein, the interaction of the candidate compound with the polypeptide can be analyzed by well known methods of detecting protein-protein interactions. . Such assays include conventional methods such as crosslinking, coimmunoprecipitation, and copurification via gradient or chromatography columns. Protein-protein interactions are also described in Fields and co-workers (Fields and Song, Nature (London) , 340: 245-246 (1989); Chien et al., Proc . Natl . Acad . Sci . USA , 88 : 9578-9582 (1991) as published by Chevray and Nathans, Proc . Natl . Acad . Sci . USA , 89: 5789-5793 (1991), can be used to monitor using the yeast-based genetic system described. Many transcriptional activators, such as yeast GAL4, consist of two physically distinct modular domains, one acting as a DNA-binding domain and the other as a transcription-active domain. The yeast expression system described in this publication (commonly referred to as "2-hybrid system") takes advantage of this property and uses a 2-hybrid protein, one of which is that the target protein is fused with the DNA-binding domain of GAL4. The other is that the candidate active protein is fused with the active domain. The expression of the GAL1-lacZ reporter gene under the control of the GAL4-activation promoter depends on the reconstitution of GAL4 activity through protein-protein interactions. Colonies comprising the interacting polypeptide are detected using a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER®), which confirms protein-protein interactions between two specific proteins using 2-hybrid technology, can be purchased from Clontech. In addition, the system can be extended to map protein domains involved in specific protein interactions, as well as to pinpoint the location of amino acid residues critical for such interactions.

In the following way, compounds which interfere with the interaction of genes encoding TAT polypeptides identified herein with other intracellular or extracellular components can be tested. A reaction mixture comprising the product of the gene encoding the TAT polypeptide and the intracellular or extracellular component was prepared under conditions and times that allow interaction and binding of the two products. To test the ability of the candidate compound to inhibit binding, the reaction was performed in the presence and absence of the test compound. In addition, placebo may be added to a third reaction mixture that serves as a positive control. Binding (conjugate formation) between test compounds present in the mixture and intracellular or extracellular components was monitored as described above. In the control reactants, the conjugate was formed, but the absence of the conjugate in the reaction mixture comprising the test compound indicates that the test compound interferes with the interaction of the test compound with its reaction counterpart.

To analyze the antagonist, TAT polypeptide could be added to the cells with the compound to screen for specific activity, and the ability of the compound to inhibit the desired activity in the presence of the TAT polypeptide indicated that the compound was an antagonist to the TAT polypeptide. Alternatively, the antagonist could be detected by binding the TAT polypeptide and potential antagonist with a membrane-bound TAT polypeptide receptor or recombinant receptor under conditions appropriate for the competitive inhibition assay. TAT polypeptides can be labeled with radioactive isotopes, for example, to determine the effect of potential antagonists using the number of TAT polypeptide molecules that bind to the receptor. Several methods known to those skilled in the art, such as ligand panning and FACS classification, see Colingan et al., Current Protocols in Immun . , 1 (2): Chapter 5 (1991)] identified genes encoding receptors. Preferably, expression cloning is used, wherein the polyadenylation RNA is prepared from cells that are responsive to the TAT polypeptide, and the cDNA library generated from the RNA is divided into pools and non-reactive to the TAT polypeptide or It is used to transfect other cells. Transfected cells that proliferate on glass slides are exposed to labeled TAT polypeptide. TAT polypeptides can be labeled by a number of methods, including the introduction of recognition sites for iodide or site-specific protein kinases. After fixation and incubation, the slides were analyzed by radiograph. Positive pools were identified to prepare sub-pools and retransfected using interactive sub-pooling and rescreening methods, resulting in a single clone encoding the putative receptor.

As an alternative method of identifying receptors, labeled TAT polypeptides could be photoaffinityly linked with extract samples expressing cell membranes or receptor molecules. The crosslinked material was analyzed by PAGE and exposed to X-ray film. Label conjugates containing receptors can be cleaved and digested into peptide fragments for protein micro-sequencing. By designing a set of degenerate oligonucleotide probes using amino acid sequences obtained from micro-sequencing, one could screen cDNA libraries and identify genes encoding putative receptors.

In another antagonist assay, mammalian cell or membrane samples expressing receptors in the presence of candidate compounds were incubated with labeled TAT polypeptide. Thereafter, the ability of the compound to enhance or block the interaction could be measured.

More specific examples of potential antagonists include oligonucleotides that bind to fusions of immunoglobulins with TAT polypeptides, and in particular poly- and monoclonal antibodies, and antibody fragments, single-chain antibodies, anti-genic antibodies, and such antibodies or Chimeric or humanized forms of fragments thereof, and antibodies, including human antibodies and antibody fragments. Alternatively, the potential antagonist may be a modified form of a TAT polypeptide that recognizes but does not affect closely related proteins such as receptors and thus competitively inhibits the action of the TAT polypeptide.

Another potential TAT polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology, where the antisense RNA or DNA molecule acts to directly block translation of the mRNA by hybridizing with the target mRNA to interfere with protein translation. Antisense techniques can be used to regulate gene expression via triple helix formation or antisense DNA or RNA, all of which are based on the binding of polynucleotides to DNA or RNA. For example, the 5 'coding portion of a polynucleotide sequence encoding a mature TAT polypeptide herein is used to design antisense RNA oligonucleotides of about 10 to 40 base pairs in length. DNA oligonucleotides are designed to be complementary to gene regions involved in transcription [Lee et al., Nucl . Acids Res. , 6: 3073 (1979); Cooney et al, Science , 241: 456 (1988); see Dervan et al., Science , 251: 1360 (1991)). Antisense RNA oligonucleotides hybridized with their mRNAs in vivo to block translation of mRNA molecules to TAT polypeptides [antisense-Okano, Neurochem . 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL, 1988). In addition, the production of TAT polypeptides can be inhibited by delivering the oligonucleotides described above to cells to express antisense RNA or DNA in vivo. If antisense DNA is used, oligodeoxyribonucleotides derived from transcription-initiation sites such as between about -10 and +10 positions of the target gene nucleotide sequence are preferred.

Potential antagonists include small molecules that bind to the active site, receptor binding site, or growth factor or other related binding site of the TAT polypeptide to block normal biological activity of the TAT polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic bipeptidyl organic or inorganic compounds.

Ribozymes are enzyme RNA molecules that can catalyze specific cleavage of RNA. Ribozymes act by sequence-specific hybridization with complementary target RNAs followed by cleavage by endonucleases. Known techniques have been able to identify specific ribozyme cleavage sites within potential RNA targets. For further details see, eg, Rossi, Current Biology , 4: 469-471 (1994) and PCT publication No. WO 97/33551 (published September 18, 1997).

The nucleic acid molecule in the triple helix form used to inhibit transcription must be single-stranded and consist of deoxynucleotides. The base composition of such oligonucleotides is designed to facilitate triple helix formation through Hoogsteen base-pairing rules, which typically require significant size purine or pyrimidine stretch in one of the double chains. For more details, see, eg, PCT publication No. WO 97/33551, supra.

Such small molecules may be identified by any one or more of the screening assays described above and / or by any other screening technique well known to those skilled in the art.

TAT polypeptide encoding nucleic acids isolated herein can be used to recombinantly produce anti-TAT polypeptides using techniques well known in the art and described herein. That is, the resulting TAT polypeptides are well known in the art and can be used to produce anti-TAT antibodies by the techniques described herein.

To treat a variety of diseases, antibodies that specifically bind the TAT polypeptides identified herein and other molecules identified by the screening assays described above can be administered in the form of pharmaceutical compositions.

Internalized antibodies are preferred if the TAT polypeptide is intracellular and all of the antibody is used as an inhibitor. However, lipofection or liposomes can also be used to deliver the antibody or antibody fragment intracellularly. If antibody fragments are used, the least inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable-region sequences of an antibody, peptide molecules can be designed that have the ability to bind a target protein sequence. Such peptides can be chemically synthesized and / or prepared by recombinant DNA techniques (eg, Marasco et al., Proc . Natl. Acad . Sci . USA , 90: 7889-7893 (1993)). Reference].

In addition, the formulations herein may comprise one or more active compounds, preferably compounds with complementary activities that do not adversely affect each other, as necessary depending on the particular condition to be treated. Alternatively, or in addition, the composition may include substances that enhance their function, such as cytotoxic substances, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are suitably formulated in amounts that are effective for the purpose intended.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

All patents and references cited herein are hereby incorporated by reference in their entirety.

Example  One: GEPIS The TAT polypeptide and / or encoding nucleic acid by means of

The desired EST sequence was confirmed by GEPIS by searching the expressed sequence tag (EST) DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.). Gene expression profiling in silico (GEPIS) is Genentech, Inc. Developed by Genentech, Inc., it is a bioinformatics tool that characterizes genes of interest as novel cancer therapeutic targets. GEPIS uses large amounts of EST sequence and library information to determine gene expression profiles. GEPIS can determine the expression profile of a gene based on its number of occurrences and its proportional correlation in the EST database, and in a rigorous and statistically meaningful manner, the LIFESEQ® EST correlation database and Genentech proprietary information It works by integrating. In this example, GEPIS was used to identify and cross-check new tumor antigens, but GEPIS can be arranged to perform very specific assays or extensive screening tasks. In initial screening, GEPIS was used to identify EST sequences from the LIFESEQ® database that correlated with expression in specific tissues or subject tissues (often subject tumor tissues). The EST sequence identified in this initial screening (or consensus sequence formed by arranging a number of related overlapping EST sequences obtained from initial screening) was then screened to confirm the presence of one or more transmembrane domains in the encoded protein. Finally, GEPIS was used to form complete tissue expression profiles for various subject sequences. Using this type of screening bioinformatics, various TAT polypeptides (and their coding nucleic acid molecules) have been found to be significantly overexpressed in certain types of cancer or some cancers compared to other cancers and / or normal noncancerous tissues. Evaluation of GEPIS hits is based on several criteria, including, for example, tissue specificity, tumor specificity and expression levels in normal essential and / or normal proliferating tissue. The tissue expression profile of the molecule as determined by GEPIS is then significantly upregulated and optionally normal required and / or high tissue expression and expression in a particular tumor or tumors relative to other tumor (s) and / or normal tissues. A series of molecules is demonstrated demonstrating relatively low expression in normal proliferating tissue. The molecules listed below are themselves excellent polypeptide targets for the diagnosis and treatment of mammalian cancer.

molecule Site where expression is upregulated comparison target DNA71290-1630 (TAT169) Breast tumor Normal breast tissue DNA71290-1630 (TAT169) Lung tumor Normal lung tissue DNA71290-1630 (TAT169) Liver tumor Normal liver tissue DNA71290-1630 (TAT169) Stomach tumor Normal stomach tissue DNA76393-1664 (TAT170) Breast tumor Breeding breast tissue DNA76393-1664 (TAT170) Colon tumor Normal colon tissue DNA76393-1664 (TAT170) Lung tumor Normal lung tissue DNA76393-1664 (TAT170) Kidney tumor Normal kidney tissue DNA76393-1664 (TAT170) Liver tumor Normal liver tissue DNA76393-1664 (TAT170) Ovarian Tumor Normal ovarian tissue DNA76393-1664 (TAT170) Pancreatic tumor Normal pancreas tissue DNA76393-1664 (TAT170) Prostate tumor Normal prostate tissue DNA76393-1664 (TAT170) Rectal tumor Normal rectal tissue DNA76393-1664 (TAT170) Brain tumor Normal brain tissue DNA76393-1664 (TAT170) Stomach tumor Normal stomach tissue DNA76393-1664 (TAT170) Uterine Tumor Normal uterine tissue DNA53971-1359 (TAT171) Prostate tumor Normal prostate tissue DNA53971-1359 (TAT171) Pancreatic tumor Normal pancreas tissue DNA53971-1359 (TAT171) Skin tumors Normal skin tissue DNA53971-1359 (TAT171) Breast tumor Normal breast tissue DNA53971-1359 (TAT171) Colon tumor Normal colon tissue DNA53971-1359 (TAT171) Kidney tumor Normal kidney tissue DNA53971-1359 (TAT171) Liver tumor Normal liver tissue DNA53971-1359 (TAT171) Stomach tumor Normal stomach tissue DNA53971-1359 (TAT171) Neuroendocrine Tumors Normal Neuroendocrine Tissue DNA53971-1359 (TAT171) Nervous system tumor Normal nervous system tissue DNA53971-1359 (TAT171) Thymic tumors Normal thymus tissue DNA56439-1376 (TAT172) Uterine Tumor Normal uterine tissue DNA56439-1376 (TAT172) Ovarian Tumor Normal ovarian tissue DNA56439-1376 (TAT172) Breast tumor Normal breast tissue DNA56439-1376 (TAT172) Colon tumor Normal colon tissue DNA56439-1376 (TAT172) Kidney tumor Normal kidney tissue DNA56439-1376 (TAT172) Pancreatic tumor Normal pancreas tissue DNA56439-1376 (TAT172) Prostate tumor Normal prostate tissue DNA56439-1376 (TAT172) Skin tumors Normal skin tissue DNA56439-1376 (TAT172) Liver tumor Normal liver tissue DNA56439-1376 (TAT172) Pituitary Tumors Normal pituitary tissue DNA56439-1376 (TAT172) Nervous system tumor Normal nervous system tissue

Example  2: Geneexpress  Tissue expression using (registered trademark) Profiling

A proprietary database containing gene expression information in an attempt to identify polypeptides (and their coding nucleic acids) whose expression is significantly upregulated in certain subject tumor tissues relative to other tumor (s) and / or normal tissues (GeneExpress ( Trademark), Gene Logic Inc., Gaithersburg, MD). Specifically, the software obtained from Gene Logic Inc., Gaithersburg, MD is used in conjunction with the GeneExpress® database or proprietary software developed by Genentech, Inc. Analysis of the GeneExpress® database was performed with the TM database. Evaluation of positive hits in this assay was based on several criteria including, for example, tissue specificity, tumor specificity and expression levels in normal essential and / or normal proliferating tissue. The following are tissue uptake profiles of molecules determined from analysis of the GeneExpress® database, with significant upregulation and optionally high tissue expression and expression in certain tumors or tumors relative to other tumor (s) and / or normal tissues. A series of molecules is demonstrated demonstrating relatively low expression in normal essential and / or normal proliferative tissue. The molecules listed below are themselves excellent polypeptide targets for the diagnosis and treatment of mammalian cancer.

molecule Site where expression is upregulated comparison target Normal tissue Tumor tissue DNA71290-1630 (TAT169) Breast tumor breast Stomach tumor top Lung tumor lungs Liver tumor liver DNA76393-1664 (TAT170) Breast tumor breast Colon tumor colon Lung tumor lungs Liver tumor liver Stomach tumor top Kidney tumor kidney Pancreatic tumor Pancreas Prostate tumor prostate Uterine Tumor Womb Rectal tumor rectal Ovarian Tumor ovary Brain tumor brain DNA53971-1359 (TAT171) Breast tumor breast Colon tumor colon Liver tumor liver Stomach tumor top Kidney tumor kidney Pancreatic tumor Pancreas Skin tumors skin Neuroendocrine Tumors Neuroendocrine system Nervous system tumor Nervous system Prostate tumor prostate Thymic tumors Thymus DNA56439-1376 (TAT172) Breast tumor breast Colon tumor colon Liver tumor liver Kidney tumor kidney Pancreatic tumor Pancreas Skin tumors skin Pituitary Tumors pituitary Nervous system tumor Nervous system Prostate tumor prostate Uterine Tumor Womb Ovarian Tumor ovary

molecule Site where expression is upregulated comparison target Normal tissue Tumor tissue DNA64852-1589 (TAT173) Breast tumor breast cervix Bone tumor goal Colon cervix esophagus Colon Heart esophagus kidney Heart liver kidney lungs liver Womb lungs ovary Womb Pancreas ovary prostate Pancreas skin prostate Intestine skin spleen Intestine top spleen testicle top bladder testicle bladder

Example  3: Hybridization As a probe  Use of TAT

The following method describes the use of hybridization probes of nucleotide sequences encoding TAT, ie the use of diagnosing tumors in mammals.

DNA comprising a coding sequence of full length or mature TAT was used as a probe for screening homologous DNA (eg, encoding naturally occurring variants of TAT) in human tissue cDNA libraries or human tissue genome libraries.

Hybridization and washing of the filters comprising these library DNAs were performed under the following high stringency conditions: The radiolabeled TAT induction probe was subjected to 50% formamide, 5 x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate. Hybridization was performed at 42 ° C. (pH 6.8), 2 × Denhardt's solution, and a solution of 10% dextran sulfate for 20 hours. The filter was washed in an aqueous solution of 0.1 x SSC and 0.1% SDS at 42 ° C.

The DNA encoding the full-length native sequence TAT and the DNA having the desired sequence identity can then be identified by standard methods known in the art.

Example  4: Lee. In Coli  Expression of TAT

In this embodiment, Recombinant expression in E. coli shows a method for producing aglycosylated forms of TAT.

First, DNA sequences encoding TAT were amplified using selected PCR primers. The primer must include a restriction site corresponding to the restriction site on the selected expression vector. Several expression vectors can be used. Examples of suitable vectors include pBR322, which includes ampicillin and tetracycline resistance genes. From coli; Bolivar et al, Gene, 2: . It is 95 (1977). The vector was digested with restriction enzymes and dephosphorylated. PCR amplified sequences were then ligated to the vector. The vector is preferably a sequence encoding an antibiotic resistance gene, a trp promoter, a poly-His leader (including the first six STII codons, a poly-His sequence and an enterokinase cleavage site), a TAT coding region, a lambda transcription terminator and an argU gene. Will include.

Subsequently, E. coli was selected using the ligation mixture, using the method described in Sambrook et al., Supra. E. coli strains were transformed. After identifying transformants that can grow on LB plates, colonies with antibiotic resistance were selected. Plasmid DNA was isolated and confirmed using restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. This overnight culture can then be used for larger inoculations. The cells are then grown to the desired optical density while the expression promoter is running.

After culturing the cells for several hours or more, the cells can be recovered by centrifugation. After dissolving the cell pellet obtained by centrifugation using various reagents known in the art, the dissolved TAT protein can be purified using a metal chelating column under conditions in which the protein is strongly bound.

TAT can be obtained using the following process. In E. coli can be expressed in the form of a poly-His tag added. DNA encoding TAT is initially amplified using selected PCR primers. Primers include restriction enzyme sites corresponding to restriction enzyme sites on selected expression vectors and other useful sequences that provide efficient and reliable initiation of translation, rapid purification on metal chelate columns, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequence was then ligated to the expression vector. E. coli host (strain 52 (W3110 fuhA (tonA) lon galE rpoHts (htpRts) clpP (lacIq)) was used to transform the transformants were first OD in LB medium containing 50 mg / mL carbenicillin. Shake culture was carried out at 30 ° C. until ₆₀₀ reached 3 to 5. The cultures were then cultured with CRAP medium (3.57 g (NH ₄ ) ₂ SO ₄ in 0.7 mL water, 0.71 g sodium citrate.2H ₂ O, 1.07 g KCl. , 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF, also prepared by mixing 110 mM MPOS, pH 7.3, 0.55% (w / v) glucose and 7 mM MgSO ₄ ). And shake culture for about 20 to 30 hours at 30 ° C. Samples were taken to confirm expression by SDS-PAGE analysis and the cells were pelleted by centrifugation of the bulk culture cells, frozen until the pellet was purified and refolded. .

E. obtained from 0.5-1 L fermentation broth. E. coli paste (6-10 g pellets) was resuspended in 10-fold volume (w / v) of 7 M guanidine, 20 mM Tris (pH 8) buffer. Solid sodium sulfite and sodium sationate were added to final concentrations of 0.1 M and 0.02 M, respectively, and the solution stirred at 4 ° C. overnight. At this stage, sulfite produced a denatured protein with all cysteine residues blocked. This solution was centrifuged for 30 minutes at 40,000 rpm in a Beckman ultracentrifuge. The supernatant was diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and clarified by filtration with a 0.22 micron filter. The clarified extracts were loaded onto a 5 mL Qiagen Ni-NTA metal chelate column equilibrated with metal chelate column buffer. The column was washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein was eluted with a buffer containing 250 mM imidazole. Fractions containing the desired protein were collected and stored at 4 ° C. Protein concentration was measured at 280 nm absorbance using the extinction coefficient calculated according to the amino acid sequence.

The sample was slowly diluted with freshly prepared refolding buffer consisting of 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA to refold the protein. The refolding volume was chosen to give a final protein concentration of 50-100 μg / mL. The refolding solution was gently stirred at 4 ° C. for 12-36 hours. The refolding reaction was stopped by adding TFA so that the final concentration was 0.4% (about pH 3). Before further purification of the protein, the solution was filtered through a 0.22 micron filter and acetonitrile was added to a final concentration of 2-10%. The refolded protein was subjected to Poros R1 / H reversed phase column chromatography using 0.1% TFA transfer buffer eluting with an acetonitrile concentration gradient of 10-80%. Aliquots of fractions showing A280 absorbance were analyzed on SDS polyacrylamide gels to recover fractions containing homogeneously refolded proteins. In general, since the refolded protein is most closely in contact with the hydrophobic inner portion that is protected from interaction with the reverse phase resin, a properly refolded protein form in most proteins elutes at the lowest concentration of acetonitrile. Aggregated proteins are usually eluted at high acetonitrile concentrations. The reverse phase step not only separates the misfolded form of protein from the desired form of protein but also removes endotoxins from the sample.

Fractions containing the folded TAT polypeptide were pooled to gently remove the acetonitrile by gently applying a stream of nitrogen to the solution. Proteins were formulated with 20 mM HEPES (pH 6.8) containing 0.14 M sodium chloride and 4% mannitol by gel filtration using dialysis or sterile filtered G25 Superfine (Pharmacia) resin equilibrated with formulation buffer.

These technique (s) were used to successfully express and purify the specific TAT polypeptides disclosed herein.

Example  5: Expression of TAT in Mammalian Cells

This example illustrates a method for producing a potential glycosylated form of TAT by recombinant expression in mammalian cells.

Vector pRK5 (see EP 307,247, published March 15, 1989) was used as the expression vector. Optionally, TAT DNA was ligated to pRK5 using the selected restriction enzyme using the ligation method described by Sambrook et al., To insert TAT DNA. The production vector was called pRK5-TAT, respectively.

In an embodiment, 293 cells can be selected as host cells. Human 293 cells (ATCC CCL 1573) were cultured onto tissue culture plates in medium such as fetal calf serum and optionally DMEM supplemented with nutrients and / or antibiotics. About 10 μg of pRK5-TAT DNA is mixed with about 1 μg of DNA encoding the VA RNA gene (Thimmappaya et al., Cell , 31 : 543 (1982)) and 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA And 0.227 M CaCl ₂ . 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO ₄ was added dropwise to the mixture to form a precipitate at 25 ° C. for 10 minutes. The precipitate was suspended and added to 293 cells and left at 37 ° C. for about 4 hours. The culture medium was aspirated off and 2 mL of 20% glycerol in PBS was added for 30 seconds. The 293 cells were then washed with serum free medium, fresh medium was added and the cells were incubated for about 5 days.

About 24 hours after transfection, the culture medium was removed and replaced with medium (only) or medium containing 200 μCi / mL ³⁵ S-cysteine and 200 μCi / mL ³⁵ S-methionine. After 12 h incubation, the conditioned media was collected, concentrated on a rotary filter and loaded onto a 15% SDS gel. The treated gel can be dried and exposed to the film for a selected period of time to confirm the presence of the TAT polypeptide. Cultures containing the transfected cells were further incubated (in serum-free medium) and the medium was tested by the selected bioassay.

As another technique, see Somparyrac et al., Proc . Natl . Acad . Sci . , 12 : 7575 (1981) using the dextran sulfate method described above could temporarily introduce TAT into 293 cells. 293 cells were cultured to the highest density in a spinner flask and 700 μg of pRK5-TAT DNA was added. Cells were first centrifuged and concentrated from spinner flasks and washed with PBS. DNA-dextran precipitates were incubated on cell pellets for 4 hours. The cells were treated with 20% glycerol for 90 seconds and washed with tissue culture medium and then placed back into the spinner flask containing tissue culture medium, 5 μg / mL bovine insulin and 0.1 μg / mL bovine transferrin. After about 4 days the conditioned media was centrifuged and filtered to remove cells and debris. The sample containing the expressed TAT can then be concentrated and purified by any method selected, such as dialysis and / or column chromatography.

In other embodiments, TAT can be expressed in CHO cells. pRK5-TAT can be transfected into CHO cells using known reagents such as CaPO ₄ or DEAE dextran. As mentioned above, the cell culture can be incubated and the culture can be replaced with a medium containing radiolabels such as medium (only) or ³⁵ S-methionine. After confirming the presence of the TAT polypeptide, the culture medium can be replaced with a serum-free medium. Preferably, the culture is incubated for about 6 days and the conditioned medium is recovered. The medium containing the expressed TAT can then be concentrated and purified by any chosen method.

In addition, epitope tagged TAT can be expressed in CHO host cells. TAT can be subcloned from the pRK5 vector. Subclonal inserts can be fused to baculovirus expression vectors in the form with selected epitope tags, such as poly-His tags, by PCR. TAT inserts with poly-His tags can be subcloned into SV40 induction vectors containing selection markers such as DHFR to select stable clones. Finally, CHO cells can be transfected with SV40 induction vectors (as above). It can be labeled in the same manner as above to confirm expression. Next, by concentrating the culture medium containing the expressed poly -His tagged TAT Ni ^{2 +} - can be purified by any selected method, such as by chromatographic our chelate affinity chroma.

TAT can also be expressed in CHO and / or COS cells by transient expression processes or in CHO cells by other stable expression methods.

The following experimental methods were used to stably express in CHO cells. Proteins are expressed and / or expressed as IgG constructs (immunoadhesines) in which the coding sequence of each protein's soluble form (e.g., the domain of a cell) is fused to an IgGl constant region sequence comprising a hinge, CH2 and CH2 domains. The poly-His tag was expressed in the added form.

Following PCR amplification, each DNA was subcloned into a CHO expression vector using standard methods described in Ausubel et al., Current Protocols of Molecular Biology, Unit 3.16, Johnn Wiley and Sons (1997). CHO expression vectors are constructed to have restriction sites suitable for the 5 'and 3' of the DNA of interest so that the restriction sites of the cDNA can be conveniently shuttled. Vectors used for expression in CHO cells are described by Lucas et al., Nucl . Acids Res. 24: 9 (1774-1779 (1996)), an SV40 early promoter / enhancer was used to express cDNA and dihydrofolate reductase (DHFR) of interest Plasmids transfected with DHFR expression The stable maintenance of can be screened.

12 μg of the desired plasmid DNA was introduced into about 10 million CHO cells using commercially available transfection reagents Superfect® (Qiagen), Dosper® or Fugene® (Boehringer Mannheim). Cells were grown according to the method described by Lucas et al., Supra. About 3 × 10 ⁷ cells were frozen in ampoules according to the method described below for later incubation and production.

Ampoules containing plasmid DNA were dissolved in a water bath and vortexed and mixed. The contents were pipetted into a centrifuge tube containing 10 mL of medium and centrifuged at 1000 rpm for 5 minutes. Supernatants were aspirated off and cells were resuspended in 10 mL selection medium (0.2 μm filtered PS20 with 0.2 μm diafiltration filtered 5% fetal bovine serum). Cells were aliquoted into 100 mL spinners containing 90 mL selection medium. After 1-2 days the cells were transferred to 250 mL spinners filled with 150 mL selection medium and incubated at 37 ° C. After 2-3 days 250 mL, 500 mL and 2000 mL spinners were inoculated with 3 × 10 ⁵ cells / mL. Cell cultures were centrifuged, exchanged with fresh medium and resuspended in production medium. Any suitable CHO medium can be used, but the production medium described in US Pat. No. 5,122,469, issued June 16, 1992, can be used in practice. 3 L production spinners were inoculated to 1.2 × 10 ⁶ cells / mL. On day 0, cell number and pH were measured. On day 1, samples were taken from the spinners to initiate sparging of the filtered air. On day 2 take a sample from the spinner, change the temperature to 33 ° C. and add 30 mL of 500 g / L-glucose and 0.6 mL of 10% antifoam (eg 35% polydimethyl siloxane emulsion, Dow Corning 365 medicinal grade emulsion) It was. During the whole production period, the pH was adjusted to around 7.2 if necessary. After 10 days or by the time the viability dropped below 70%, the cell culture was recovered by centrifugation and filtered through a 0.22 μm filter. The filtrate was stored at 4 ° C. until loaded into the purification column.

For poly-His tagged constructs, proteins were purified using a Ni-NTA column (Qiagen). Imidazole was added to the conditioned medium at 5 mM concentration before purification. The conditioned medium was injected into a 6 mL Ni-NTA column equilibrated with 20 mM HEPES (pH 7.4) buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 mL / min at 4 ° C. After loading the column was washed with additional equilibration buffer and the protein was eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was then desalted in 25 mL G25 Superfine (Pharmacia) column in storage buffer (pH 6.8) containing 10 mM HEPES, 0.14 M NaCl and 4% mannitol and stored at -80 ° C.

The immunoadhesin (including Fc) construct was purified from the conditioned medium as follows. The conditioned medium was injected into a 5 mL Protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer, pH 6.8. After loading, the column was washed thoroughly with equilibration buffer and then eluted with 100 mM citric acid (pH 3.5). The eluted protein was recovered in 1 mL fractions in a tube containing 275 μl of 1 M Tris buffer (pH 9) and immediately neutralized. The highly purified protein was then removed with salt in storage buffer by the method described above in the case of poly-His tagged proteins. Homology was confirmed by SDS-polyacrylamide gel and N-terminal amino acid sequencing performed by Edman digestion.

These technique (s) were used to successfully express and purify the specific TAT polypeptides disclosed herein.

Example  6: In yeast  Expression of TAT

The following method describes recombinant expression of TAT in yeast.

First, yeast expression vectors for intracellular production or secretion of TAT were prepared from the ADH2 / GAPDH promoter. DNA encoding TAT was inserted into a suitable restriction enzyme site of the plasmid selected for intracellular expression of TAT. To secrete TAT, DNA encoding the ADH2 / GAPDH promoter, native TAT signal peptide or other mammalian signal peptide or for example yeast alpha-factor or invertase secretion signal / leader sequence and linker sequence (if required); The DNA encoding the TAT together was cloned into the selected plasmid to express TAT.

Yeast cells, for example yeast strain AB110, can be transformed with the expression plasmids described above and cultured in selected fermentation medium. Transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid, separated by SDS-PAGE and stained with Coomassie Blue.

The yeast cells can then be removed from the fermentation medium by centrifugation and the medium concentrated using a selected cartridge filter to isolate and purify the recombinant TAT. Concentrates containing TAT can be further purified using selected column chromatography resins.

These technique (s) were used to successfully express and purify the specific TAT polypeptides disclosed herein.

Example  7: With baculovirus  Expression of TAT in Infected Insect Cells

The following method describes recombinant expression of TAT in insect cells infected with baculovirus.

The coding sequence of the TAT was fused upstream of the epitope tag included in the baculovirus expression vector. The epitope tag includes a poly-His tag and an immunoglobulin tag (like the Fc region of IgG). Various plasmids can be used, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, 5 'and 3 the coding sequence of the TAT or the desired portion of the coding sequence of the TAT, e. PCR was amplified using primers complementary to the region. The 5 'primer may comprise flanking (selected) restriction enzyme sites. The product was then digested with the selected restriction enzyme and subcloned into the expression vector.

Recombinant baculovirus uses Lipofectin (Gibco-BRL) to transfer the above plasmid and BaculoGold® DNA (Pharmingen) to Spodoptera frugiperda ("Sf9") cells ( And were simultaneously transfected into ATCC CRL 1711). After 4-5 days of incubation at 28 ° C., the released virus was recovered and used for subsequent amplification. Viral infections and protein expression are described in O'Reilley et al., Baculovirus Expresstion vectors : A laboratory Manual , Oxford: Oxford University Press (1994).

Then, the expressed poly -His tagged TAT are, for example Ni ^{2 +} - can be purified as follows: to a Mato our chelate affinity chroma. Extracts were prepared from recombinant virus-infected Sf9 cells according to Rupert et al., Nature , 362 : 175-179 (1993). Briefly, Sf9 cells were washed and resuspended in sonication buffer (25 mL HEPES, pH 7.9; 12.5 mM MgCl ₂ ; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl) for 20 seconds on ice. Sonication was performed twice. The sonication was removed by centrifugation and the supernatant diluted 50-fold in loading buffer (50 mM phosphoric acid, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. The Ni ²⁺ -NTA agarose column (purchased from Quaiagen) was washed with 25 mL of water using 5 mL fill volume and equilibrated with 25 mL of loading buffer. The filtered cell extracts were loaded into the column at 0.5 mL per minute. At the start of point fraction recovery, the column was washed with loading buffer to the A ₂₈₀ baseline. Next, the column was washed with secondary wash buffer (50 mM phosphoric acid; 300 mM NaCl, 10% glycerol, pH 6.0) to elute nonspecifically bound protein. When A ₂₈₀ reached baseline again, the column was developed in gradient with 0-500 mM imidazole in secondary wash buffer. 1 mL fractions were recovered and analyzed by Western blotting using a Ni ^{2 +} -NTA (Qiagen) coupled to SDS-PAGE and silver staining or alkaline phosphatase. Fractions containing eluted His ₁₀ tagged TAT were pooled and dialyzed against loading buffer.

Alternatively, purification of IgG tagged (or Fc tagged) TAT can be performed using known chromatography techniques such as Protein A or Protein G column chromatography.

These technique (s) were used to successfully express and purify the specific TAT polypeptides disclosed herein.

Example  8: Preparation of an Antibody That Binds TAT

This example describes a method for preparing monoclonal antibodies that can specifically bind to TAT.

Techniques for producing monoclonal antibodies are known in the art and are described, for example, in Goding, supra. Immunogens that can be used include fusion proteins comprising purified TAT and cells expressing recombinant TAT on the cell surface. One skilled in the art can select immunogens without undue experimentation.

Mice, for example Balb / c, were immunized subcutaneously or intraperitoneally with 1-100 μg of TAT immunogen emulsified in Freund's complete adjuvant. Alternatively, the immunogen was emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the hind paw of the animal. Immunized mice were then boosted with additional immunogen emulsified in selected adjuvant after 10-12 days. Thereafter, mice may be boosted with further immunization injections for several weeks. Serum samples were taken periodically from mice by reverse bleeding, and the ELISA assay was tested to detect anti-TAT antibodies.

Once a suitable antibody titer is detected, a final intravenous TAT can be given to an animal "positive" for the antibody. After 3-4 days, the mice were sacrificed to recover the spleen cells. The spleen cells were then fused with P3X63AgU.1 available from selected murine myeloma cell lines, eg ATCC No. CRL 1597 (using 35% polyethylene glycol). The fusions are hybridoma cells that can be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin and thymidine) media that inhibit the proliferation of non-fusion cells, myeloma hybrids and splenocyte hybrids. Generated.

Hybridoma cells are screened by ELISA for reactivity with TAT. Methods of determining "positive" hybridoma cells that secrete the desired monoclonal antibodies against TAT are known to those of skill in the art.

Positive hybridoma cells can be injected intraperitoneally so that Balb / c mice of genetically identical lineage produce ascites containing anti-TAT monoclonal antibodies. Alternatively, the hybridoma cells can be cultured in tissue culture flasks or roller bottles. Monoclonal antibodies generated in the ascites can be purified through ammonium sulfate precipitation and subsequent gel exclusion chromatography. Alternatively, affinity chromatography can be used based on protein A or protein G binding of an antibody.

These technique (s) were used to successfully express and purify the specific TAT polypeptides disclosed herein.

Example  9: Purification of TAT Polypeptides Using Specific Antibodies

Natural or recombinant TAT polypeptides can be purified through various standard techniques in the field of protein purification. For example, pro-TAT polypeptides, mature TAT polypeptides, or pre-TAT polypeptides are purified by immunoaffinity chromatography using antibodies specific for the desired TAT polypeptide. In general, immunoaffinity columns were constructed by covalently coupling an anti-TAT polypeptide antibody to an activated chromatography resin.

Polyclonal immunoglobulins were prepared from ammonium serum by either ammonium sulfate precipitation or purification on immobilized protein A (Pharmacia LKB Biotechnology, Piscataway, NJ). Similarly, monoclonal antibodies were prepared by ammonium sulfate precipitation or chromatography on immobilized protein A from mouse ascites. Partially purified immunoglobulins were covalently attached to chromatographic resins such as CnBr-activated Sepharose ™ (SEPHAROSE) ™ (Pharmacia LKB Biotechnology). The antibody was coupled to the resin, the resin blocked and the derived resin was washed according to the manufacturer's instructions.

Such immunoaffinity columns are used for purification of TAT polypeptide by preparing fractions from cells comprising the TAT polypeptide in soluble form. This agent is derived by the addition of detergents or by the solubilization of subcellular fractions obtained by solubilization or differential centrifugation of intact cells by other methods known in the art. Alternatively, the soluble TAT polypeptide comprising the signal sequence may be secreted in a useful amount into the medium in which the cell grows.

The soluble TAT polypeptide containing agent was passed through an immunoaffinity column and the column was washed under conditions that allow preferential absorbance of the TAT polypeptide (eg, high ionic strength buffer in the presence of a detergent). The column is then eluted under conditions that disrupt antibody / TAT polypeptide binding (e.g., low pH buffers such as about pH 2-3 or high concentrations of chaotrope, e.g. urea or thiocyanate ions). TAT polypeptide was recovered.

Deposit of matter

The following materials have been deposited with the American Type Culture Collection (ATCC), Manassas University Boulevard 10801, Virginia, USA: 20110-2209:

matter ATCC  Deposit number Deposit date

DNA71290-1630 203275 September 22, 1998

DNA76393-1664 203323 October 6, 1998

DNA53971-1359 209750 April 7, 1998

DNA56439-1376 209864 May 14, 1998

DNA64852-1589 203127 August 18, 1998

These deposits were made under the provisions of the Budapest Treaty and its rules (Budapest Treaty) on the international approval of microbial deposits under the patent procedure. This ensures maintenance of the viable culture of the deposit for 30 years from the date of deposit. The deposits will be distributed from ATCC under the Convention of the Budapest Treaty under the agreement between Genentech Inc and ATCC, upon the granting of the relevant U.S. patent or the publication of a U.S. or foreign patent application (when it is first). To ensure the permanent and non-limiting distribution of the deposit progeny, and to the regulations of the US Patent and Trademark Commissioner, 35 USC §122 and the rules of the US Patent and Trademark Commissioner (including 37 CFR §1.14, in particular 886 OG 638). Progenie's sale is assured to those who decide to have rights under

The assignee of the present application agrees that upon incubation under appropriate conditions, if the culture of the deposited material is killed, lost or damaged, the material will be replaced immediately with another identical material upon notification. The sale of deposited materials should not be construed as an authorization of the governments to carry out the invention in violation of the rights granted under the patent law.

The foregoing description is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not intended to be limited to the scope of the deposited artwork as the deposited embodiments are intended as an illustration of certain aspects of the invention, and any functionally equivalent construction is within the scope of the invention. The deposit of a substance herein does not mean that the disclosure contained herein is inappropriate for carrying out any aspect, including the best mode of the invention, and is intended to limit the scope of the claims to the specific description set forth in the specification. It should not be interpreted. Also, various modifications of the present invention to those skilled in the art other than those shown and described herein above are apparent to those skilled in the art, which will be within the scope of the appended claims.

Antibodies of the invention can be used to identify polypeptides of various cells (and coding nucleic acids or fragments thereof) that are expressed at a higher degree on the surface of one or more types of cancer cells than one or more types of normal non-cancer cells. Such polypeptides are referred to herein as tumor-associated Antigenic Target polypeptides (“TAT” polypeptides), which are expected to function as effective targets for cancer therapy and diagnosis in mammals.

Attach sequence list electronic file

Claims (13)

(a) the amino acid sequence of SEQ ID NO: 10; (b) the amino acid sequence of SEQ ID NO: 10, lacking a linked signal peptide; (c) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 5; (d) an amino acid sequence encoded by the full length coding sequence of the nucleotide sequence of SEQ ID NO: 5; or (e) an amino acid sequence encoded by the full length coding sequence of the cDNA deposited with ATCC Accession No. 203127 An isolated antibody that binds to a polypeptide having. The antibody of claim 1 which is a monoclonal antibody. The antibody of claim 1 which is an antibody fragment. The antibody of claim 1, which is a chimeric or humanized antibody. The antibody of claim 1 conjugated to a growth inhibitor. The antibody of claim 1 conjugated to a cytotoxic agent. The antibody of claim 6, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes. The antibody of claim 6, wherein the cytotoxic agent is a toxin. The antibody of claim 8, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicins. The antibody of claim 8, wherein the toxin is a maytansinoid. The antibody of claim 1 produced in a bacterium. The antibody of claim 1, which is produced in CHO cells. The antibody of claim 1, wherein the antibody is detectably labeled.

Images